The Plant Cell, Vol. 13, 351–367, February 2001, www.plantcell.org © 2001 American Society of Plant Physiologists

bus

, a Bushy Arabidopsis

CYP79F1

Knockout Mutant with Abolished Synthesis of Short-Chain Aliphatic Glucosinolates

Birgit Reintanz,

a,e

Michaela Lehnen,

a

Michael Reichelt,

b

Jonathan Gershenzon,

b

Marius Kowalczyk,

c

Goran Sandberg,

c

Matthias Godde,

a

Rainer Uhl,

d

and Klaus Palme

a,1

a

Max-Delbrück-Laboratorium, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany

b

Max-Planck-Institut für Chemische Ökologie, Carl-Zeiss-Promenade 10, D-07745 Jena, Germany

c

Swedish University of Agricultural Science, Petrus Laestadius vag, SE-901 83 Umeå, Sweden

d

BioImaging Zentrum der Ludwig Maximilians Universität München, D-82152 Martinsried, Germany

e

Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany

A new mutant of Arabidopsis designated

bus1-1

(for

bushy

), which exhibited a bushy phenotype with crinkled leavesand retarded vascularization, was characterized. The phenotype was caused by an

En-1

insertion in the gene

CYP79F1

.The deduced protein belongs to the cytochrome P450 superfamily. Because members of the CYP79 subfamily are be-lieved to catalyze the oxidation of amino acids to aldoximes, the initial step in glucosinolate biosynthesis, we analyzedthe level of glucosinolates in a CYP79F1 null mutant (

bus1-1f

) and in an overexpressing plant. Short-chain glucosino-lates derived from methionine were completely lacking in the null mutant and showed increased levels in the overex-pressing plant, indicating that CYP79F1 uses short-chain methionine derivatives as substrates. In addition, theconcentrations of indole-3-ylmethyl-glucosinolate and the content of the auxin indole-3-acetic acid and its precursorindole-3-acetonitrile were increased in the

bus1-1f

mutant. Our results demonstrate for the first time that the formationof glucosinolates derived from methionine is mediated by CYP79F1 and that knocking out this cytochrome P450 hasprofound effects on plant growth and development.

INTRODUCTION

Glucosinolates (mustard oil glycosides) are plant secondarymetabolites that are found almost exclusively in plants ofthe order Capparales (Rodman, et al., 1996; Selmar, 1999).These compounds are sulfonated thioglucosides, whichare stored in the vacuole and are hydrolyzed upon celldamage by thioglucosidases known as myrosinases (Bonesand Rossiter, 1996). The resulting aglycones rearrange toform a variety of products that are toxins and deterrents tomany pathogens and herbivores and that are believed to servein plant defense (Louda and Mole, 1991). On the basis oftheir toxic properties and pungent taste, glucosinolates areoften classified as antinutritional compounds. For example,rapeseed meal, a by-product of oil extraction from crushedrapeseed, is an excellent feed concentrate whose nutritionalvalue is impaired by a high concentration of glucosinolates.However, glucosinolates are also of substantial benefit to hu-mans as flavoring agents in a variety of cruciferous vegeta-bles (e.g., broccoli and cauliflower) and spices (e.g., mustard).

Furthermore, the consumption of such vegetables seemsto reduce the risk of developing cancer (Jongen, 1996;Verhoeven et al., 1997). A hydrolytic degradation productof the methionine-derived 4-methylsulfinylbutyl (4MSOB)glucosinolate, 4MSOB isothiocyanate, was found to be apotent anticarcinogen (Zhang et al., 1992). Therefore, gluco-sinolates are considered to be excellent candidates for usein creating “functional food” designed to reduce the risk ofcancer.

Although significant progress in understanding glucosino-late biosynthesis has been made in recent years, the char-acteristics of the enzymes involved are still imperfectlyknown (Halkier and Du, 1997; Halkier, 1999). The first step inglucosinolate biosynthesis appears to be the oxidation ofamino acids to their corresponding aldoximes, which isanalogous to the first step in cyanogenic glycoside forma-tion. After donation of a thiol moiety (probably from cys-teine), sequential transfer of glucose and sulfate residuescompletes the basic skeleton. At present, there is consider-able controversy surrounding the nature of the enzymes thatcatalyze aldoxime formation. For example, in rapeseed, oxi-dation of phenylalanine to its corresponding aldoxime has beenattributed to a flavin-containing monooxygenase (Bennett etal., 1993), whereas in white mustard, aldoxime formation

1

To whom correspondence should be addressed. E-mail palme@mpiz-koeln.mpg.de; fax 49-221-5062-613.

352 The Plant Cell

Figure 1.

Phenotype of the

bus1-1

Mutant.

bus

, a

CYP79F1

Knockout Mutant 353

from tyrosine appears to be mediated by a cytochromeP450 monooxygenase (Du et al., 1995). Furthermore, per-oxidases have been proposed to be involved in aldoximeproduction from tryptophan in Chinese cabbage (Ludwig-Müller and Hilgenberg, 1988).

The model plant Arabidopsis, belonging to the Brassi-caceae family in the order Capparales, contains more than30 different glucosinolates (Hogge et al., 1988; Haughn etal., 1991). Therefore, this species is well suited to the exam-ination of glucosinolate biosynthesis and its physiologicalroles. The glucosinolates in Arabidopsis originate principallyfrom phenylalanine, tryptophan, and chain-elongated me-thionine derivatives. To date, three genes involved in al-doxime formation have been cloned from this species (Hullet al., 2000; Mikkelsen et al

.

, 2000; Wittstock and Halkier,2000). The deduced proteins (CYP79A2, CYP79B2, andCYP79B3) all belong to the P450 superfamily. Heterologousexpression studies have shown that these enzymes are ableto convert phenylalanine (CYP79A2) and tryptophan (CYP79B2and CYP79B3) to their corresponding aldoximes. In addi-tion, transgenic Arabidopsis plants overexpressing CYP79A2contained higher concentrations of benzylglucosinolate thandid wild-type plants, indicating that CYP79A2 uses phenyl-alanine as substrate in vivo (Wittstock and Halkier, 2000).

Here, we describe the isolation and characterization ofthe Arabidopsis mutant

bus1-1

(for

bushy

), a bushy, semi-sterile plant with crinkled leaves. The phenotype wascaused by an

En-1

insertion in the gene

CYP79F1

encod-ing a cytochrome P450 monooxygenase. The expressionof CYP79F1 in apical plant parts was restricted to the vas-cular tissues. Analysis of a null mutant (

bus1-1f

) revealedthe complete lack of short-chain methionine-derived glu-cosinolates, demonstrating that CYP79F1 is involved in thebiosynthesis of aliphatic glucosinolates. Furthermore, over-expression of CYP79F1 in Arabidopsis led to an increasedlevel of short-chain methionine-derived glucosinolates. Ad-ditionally, the

bus1-1f

mutant contained an increased levelof indole-3-ylmethyl-glucosinolate together with increasedindole-3-acetic acid (IAA) and indole-3-acetonitrile (IAN)levels, which could be responsible for the observed devel-opmental phenotypes. These findings might indicate thatalterations in the metabolism of methionine-derived gluco-

sinolates may directly or indirectly affect auxin metabolismand plant morphogenesis.

RESULTS

Phenotype Analysis of an

En-1–

Induced

bus1-1

Mutant

A collection of Arabidopsis plants mutagenized with themaize

En-1

transposon (Wisman et al., 1998) was screenedfor plants with changed shoot growth at maturity. The

bus1-1

mutant was chosen for further characterization because ofthe extremely bushy phenotype (Figure 1A). Segregationanalysis revealed that the parent plant (collection number4AAJ108) was heterozygous and that the phenotype wascaused by a recessive mutation.

Under normal greenhouse conditions, the

bus1-1

mutantwas characterized by (1) small crinkled leaves, (2) early for-mation of secondary rosette leaves, (3) semisterility, and (4)an enhanced number of lateral shoots. The first differencesnoted between the mutant and wild-type plants were evi-dent in the cotyledons of 5-day-old seedlings. At this stage,the vascularization of cotyledons was restricted to the pri-mary vein, whereas wild-type plants had already formedsecondary veins. Retardation of vascular strand formationoccurred at all developmental stages but was most pro-nounced in fully developed rosette leaves (Figure 1F). Thedefective reticularization of the vascular system might bethe reason for the observed leaf shape. Juvenile rosetteleaves were strongly curled upward, and the two halves ofthe blade had the appearance of being hinged along themidrib, resembling the leaves of a Venus flytrap. Later in de-velopment, the leaves expanded, although the margins re-mained curled. Adult leaves were crinkled (Figure 1E).Rosettes of the mutant were smaller compared with those ofthe wild type (Figure 1C). Furthermore, secondary leavesthat developed in the rosette leaf axils were larger and morenumerous than those in the wild type.

The onset of the main inflorescence and the floweringtime were not affected in the mutant (Figure 1B). Analyses offlower morphology revealed that the number of floral organs

Figure 1.

(continued).

(A)

to

(D)

show

bus1-1

at left and wild type at right.

(A)

The mature

bus1-1

mutant exhibits a small bushy phenotype.

(B)

At flowering time, the first side shoot reaches the height of the main inflorescence (5 weeks old).

(C)

Under normal greenhouse conditions, the

bus1-1

mutant has smaller rosette leaves (3 weeks old).

(D)

Under short-day conditions, the rosette leaves are normal in size (6 weeks old).

(E)

Fully developed rosette leaves of

bus1-1

are crinkled.

(F)

bus1-1

leaves (bottom) have fewer veins than do wild-type leaves (top).

(G)

The

bus1-1

mutant is semisterile, with siliques that vary in size or do not develop at all.

(H)

Most mutant pistils are already visible when floral buds are still closed, so the anthers cannot reach the stigmas during dehiscence.

354 The Plant Cell

varied strongly in the mutant. Flowers had three to four se-pals (the wild type had four), one to five petals (wild type,four), and three to five stamens (wild type, six). No differ-ences could be observed in the sizes of these organs. Thenumber of pistils was not affected, but their length variedstrongly. Many pistils were already visible at a stage atwhich floral buds were still closed (Figure 1H). A variableamount of pollen was released on the stigma, depending onwhether the anthers could reach the stigma during dehis-cence. As a consequence, fertilization was often reduced, anddeveloping siliques of

bus1-1

were variable in size and seedcontent and sometimes did not develop at all (Figure 1G).

At the time of flowering, neither the length of the main inflo-rescence nor the number of lateral shoots differed betweenthe

bus1-1

mutant and the wild type. However, differenceswere visible in the length of the lateral shoots. In the mutant,the shoot closest to the shoot apex had already reached theheight of the main inflorescence (Figure 1B). After flowers be-gan to open, the main inflorescence of the mutant stoppedgrowing or grew much slower compared with that of the wildtype. Additionally, secondary inflorescences that emergedfrom the rosette leaf axils were increased in number andlength. The secondary inflorescences also stopped growingafter floral transition. The reduced growth, together with theenhanced secondary shoot formation, caused the bushy phe-notype observed at maturity (Figure 1A).

All of these characteristics of the

bus1-1

phenotype werealso observed under short-day conditions, except for thedifference in leaf size. Under these conditions, the

bus1-1

mutant developed leaves that were normal in size (Figure1D). When the root development of seedlings grown in vitro

was analyzed, no differences in the length or the number oflateral roots were noted.

Molecular Characterization of Different

bus

Mutants

To determine if the gene of interest in the

bus1-1

mutantwas still tagged by an

En-1

insertion, we screened offspringpopulations for plants that had reverted to the wild-typephenotype. Germinal reversion was detected at high fre-quencies (20 to 70%), indicating that the observed pheno-type was caused by transposon insertion. Nevertheless, dueto the high

En-1

copy number (10 to 15), several methodsused to isolate the corresponding flanking region failed. Toreduce the number of

En-1

insertions in the genome, themutant was crossed to the wild type. In the F2 generation,different lines segregating for the

bus1-1

phenotype wereidentified, but DNA gel blot analysis still revealed a copynumber corresponding to at least 10

En-1

insertions. Addi-tionally, germinal reversion could not be observed in the off-spring of the F2–

bus1-1

mutants, indicating that excision of

En-1

during the crossing procedure probably caused a sta-ble footprint mutation. This footprint was confirmed later(see below).

To identify new mutants with fewer

En-1

insertions, we re-screened the Arabidopsis

En-1

collection for plants exhibit-ing the characteristic

bus

phenotype. Three additionalindependent mutants were found, and segregation analysisof the parent plants (collection numbers 4L9-1, 6N18, and6AAQ19) revealed that all of them were heterozygous. Wechose plant 4L9-1, which had approximately four

En-1

in-

Figure 2. Genomic Organization of CYP79F1 and CYP79F2.

The coding regions of both genes comprise three exons (white boxes). Translation initiation sites are indicated by arrows, and intergenic regionsthat have been used for GUS expression studies are indicated by dashed lines. In both identified bus alleles (bus1-1 and bus1-2), the En-1 trans-poson is located in the second exon, although in different orientations (arrows). Insertion of En-1 in bus1-1 caused a 3-bp target duplication(AGT in boldface). The stable footprint mutant bus1-1f contains four additional bases, causing a frameshift (changed bases and amino acids areshown in boldface).

bus

, a

CYP79F1

Knockout Mutant 355

sertions, for further characterization and designated the mu-tant allele

bus1-2

. Combined phenotypic and geneticanalysis of the 4L9-1 progeny revealed an

En-1

fragmentcosegregating with the mutant phenotype (see Methods).The flanking region of this

En-1

insertion was isolated by apolymerase chain reaction (PCR)–based strategy and se-quenced. Database searches identified this sequence in thebacterial artificial chromosome (BAC) clone F309 (GenBankaccession number AC006341). The

En-1

transposon wasfound to be inserted in gene

F309.21

located on chromo-some 1 (Figure 2).

On the basis of this gene sequence, we reanalyzed theoriginal

bus1-1

mutant. PCR reactions with gene-specificprimers in combination with different

En-1

primers revealedthat

bus1-1

also contained an

En-1

insertion in gene

F309.21

(Figure 2). This insertion could no longer be de-tected in the F2–

bus1-1

mutants derived from the crosseswith the wild type (see above). Amplification with two

F309.21

-specific primers that flank the original insertion siterevealed a sequence containing four additional bases, caus-ing a frameshift (Figure 2). This stable null mutant has beendesignated

bus1-1f

(for footprint).

Identification of CYP79F1 and CYP79F2

Detailed sequence analysis of the BAC clone F309 led to theidentification of the gene

F309.20

1.5 kb upstream of

F309.21

, with 86% homology to

F309.21

(Figure 2). On thebasis of these sequences, primers comprising the entirecoding regions were designed for reverse transcription (RT)–PCR amplification of the cDNAs. Sequence comparison re-vealed that each gene contains three exons spanning

z

2.1kb of genomic DNA (Figure 2). The predicted proteins havea calculated molecular mass of

z

62 kDa and share 89%identity at the amino acid level (Figure 3). The deducedamino acid sequences exhibited homology with conservedmotifs found in cytochrome P450 enzymes, including theproline-rich domain (consensus, [P/I]PGPX[G/P]XP) and theheme binding domain (consensus, FXXGXXXCXG) (Chapple,1998) (Figure 3). The greatest level of identity,

z

45%, wasfound to the members of the CYP79 family. According to thenomenclature for the cytochrome P450 superfamily, theproteins were thus designated CYP79F1 (gene

F309.21

)and CYP79F2 (gene

F309.20

) (http://drnelson.utmem.edu/CytochromeP450.html).

Figure 3. Amino Acid Comparison of CYP79F1 and CYP79F2.

The deduced proteins share 89% identical amino acids (black). At the N termini, the four motifs characteristic of most cytochrome P450 en-zymes are indicated. The N-terminal regions fused with GFP (named N-CYP79F1 and N-CYP79F2) are marked by arrows. The heme binding do-main is located in the C-terminal part of the proteins.

356 The Plant Cell

Subcellular Localization of CYP79F1 and CYP79F2

CYP79F1 and CYP79F2 are very similar, but significant dif-ferences occur in their extreme N-terminal regions (Figure3). Several motifs were identified in the N-terminal regionsthat are conserved in other cytochrome P450s: a short N-ter-minal signal peptide for targeting to the endoplasmic reticu-lum (ER); a stretch of hydrophobic amino acids constitutingthe transmembrane helix, which anchors the protein in theER; a positively charged domain; and a proline-rich motif(Chapple, 1998) (Figure 3). Consistent with these motifs,computer analyses of CYP79F1 and CYP79F2 suggested alocalization in the ER (program TargetP at http://www.cbs.dtu.dk/services). However, because of the observed aminoacid differences at the N termini of the two proteins, itseemed important to prove ER localization experimentally.The first 62 amino acids of either CYP79F1 or CYP79F2(named N-CYP79F1 and N-CYP79F2), which include the mo-tifs mentioned above (Figure 3), were fused to green fluores-cent protein (GFP). As controls, we used either GFP alone,which is localized to the cytoplasm, or a GFP variant that istargeted to the ER (Haselhoff et al., 1997). All constructs wereexpressed in tobacco BY2 cells. Comparison of the fluores-cence patterns between the controls and the fusion proteinsrevealed that the GFP fusions of CYP79F1 and CYP79F2were both located in the ER (Figure 4).

Studies on the Expression of

CYP79F1

and

CYP79F2

Due to the great similarity of CYP79F1 and CYP79F2, it wasnot possible either to generate the specific DNA probesneeded for RNA gel blot analyses and in situ hybridizationstudies or to synthesize the specific peptides needed for an-tibody production and subsequent protein gel blot analysis.Therefore, we performed promoter analysis for each gene intransgenic Arabidopsis plants. Both promoters (

PCYP79F1

and

PCYP79F2

) were amplified by PCR and fused to the

uidA

reporter gene.

PCYP79F1

corresponds to the entire in-tergenic region between gene

F309.20

and

F309.21

(1.5 kb),whereas

PCYP79F2

comprises the region between gene

F309.19 and F309.20 (0.7 kb; Figure 2). The constructs weretransformed into Arabidopsis, and tissue-specific expres-sion patterns were analyzed over three generations. Histo-chemical staining revealed variations in b-glucuronidase (GUS)activity in different transgenic lines of both PCYP79F1 andPCYP79F2. Such variability is common and probably re-flects the positional effects of the insertion site on transgeneexpression. Nevertheless, all lines exhibited the same quali-tative staining pattern.

No differences in the activity patterns of the two promot-ers were found in apical plant parts. Microscopic analysis ofwhole organs revealed promoter activities exclusively in thevascular bundles of hypocotyls, leaves, stems, and maturesiliques (Figure 5A). The highest level of promoter activitywas observed in fully expanded rosette leaves, and the low-

Figure 4. Heterologous Expression in BY2 Cells.

Different GFP constructs (at top) were expressed under the controlof the cauliflower mosaic virus (CaMV) 35S promoter in tobacco BY2cells. Transformed cells were analyzed either by differential interfer-ence contrast (at bottom left) or by fluorescence microscopy (at bot-tom right).(A) Expression of N-CYP79F1 (see Figure 3) fused to GFP. Greenfluorescence was observed at the cortical and perinuclear ER. TheN-CYP79F2–GFP fusion protein exhibited the same fluorescencepattern (not shown).(B) Expression of GFP alone. Green fluorescence was observed inthe cytoplasm.(C) Expression of a GFP variant targeted to the endoplasmic reticu-lum (GFP-ER). Green fluorescence was restricted to the ER.

bus, a CYP79F1 Knockout Mutant 357

est level was seen in stems and young stem leaves. No ac-tivity was found in flowers.

Striking differences in the activity of PCYP79F1 andPCYP79F2 were observed in roots. GUS staining fromPCYP79F2 activity was found throughout the root, except inthe root tips (Figure 5B, left). Activity was restricted to the vas-cular system and was first detectable 3 days after germinationat the basal end of the main root (Figure 5B, right). In contrast,PCYP79F1 was not active in the main root (Figure 5C, left) butwas restricted to young lateral root primordia and vascular tis-sue at the basal ends of lateral roots (Figure 5C, right).

To confirm the results of the GUS expression analysis, RT-PCR was performed with gene-specific primers for CYP79F1and CYP79F2. Specificity was tested in PCR reactions withgenomic DNA, in which mispriming would have given rise toPCR products of different lengths. RT-PCR analyses with

poly(A)1 RNA from various tissues of wild-type Arabidopsisplants revealed the following features (Figure 5D): (1) tran-scription of both genes in all analyzed tissues except flowers;(2) no significant differences in transcription levels betweenCYP79F1 and CYP79F2 in shoots; and (3) a considerablylower transcription level of CYP79F1 than of CYP79F2 inroots. Together, these results were in full agreement with theresults of the GUS expression studies.

Analysis of Glucosinolate Patterns

Members of the Arabidopsis CYP79 family are believed tocatalyze the conversion of amino acids to aldoximes, thefirst step in glucosinolate formation (Bak et al., 1998). Heter-ologous expression in Escherichia coli of CYP79A2,

Figure 5. Expression Studies of CYP79F1 and CYP79F2 by GUS Staining and RT-PCR.

(A) Promoter activity of PCYP79F1 in apical plant parts. Expression in hypocotyls, leaves, stems, silique stems, and siliques (left to right) is re-stricted to the vascular tissue. PCYP79F2 exhibited the same staining pattern (not shown).(B) and (C) Differences in the expression patterns are visible in roots.(B) PCYP79F2 is active in the vascular tissue throughout the entire root (left) except for the root tips (right, top) and is first detectable in 3-day-old seedlings at the basal end of the main root (right, bottom).(C) PCYP79F1 is not active in the main root (left) and is detected only in lateral root primordia (right, top) and at the basal ends of lateral roots(right, bottom).(D) RT-PCR reactions using poly(A)1 RNA from roots (r), rosette leaves (rl), stem leaves (sl), stems (s), flowers (f), and siliques (si) with gene-spe-cific primers for CYP79F1 (F1), CYP79F2 (F2), and actin (a).

358 The Plant Cell

Figure 6. Estimation of Glucosinolate Content by HPLC Analysis.

bus, a CYP79F1 Knockout Mutant 359

CYP79B2, and CYP79B3 from Arabidopsis showed thatthese proteins are able to synthesize aldoximes from phenyl-alanine (CYP79A2) and tryptophan (CYP79B2 and CYP79B3)(Hull et al., 2000; Mikkelsen et al., 2000; Wittstock and Halkier,2000). However, given the claims for the involvement ofother enzymes in this reaction type, the role of the CYP79proteins is not fully clear. The availability of the stable foot-print mutant bus1-1f now allows assessment of whether ornot CYP79F1 is involved in glucosinolate biosynthesis.

Glucosinolate patterns in roots, leaves, and seed of bus1-1fwere compared with those of wild-type plants. HPLC analy-ses revealed that many glucosinolates derived from me-thionine were below detectable limits in bus1-1f, suggestingthat CYP79F1 is involved in the synthesis of aldoximes frommethionine (Figure 6). In fact, all of the major aliphatic glu-cosinolates of Arabidopsis appear to be derived from chain-elongated derivatives of methionine that are formed by athree-step cycle that yields an amino acid with one to sixadditional methylene groups in the side chain (Graser et al.,2000). After these methionine derivatives are converted toglucosinolates, subsequent side chain modifications (oxida-tion, loss of the methylsulfinyl group, and esterification) leadto the formation of the great variety of glucosinolates in Ara-bidopsis. In the bus1-1f mutant, all of the glucosinolates de-rived from methionine derivatives extended by one to fourmethylene residues were completely absent (Figure 6 andFigure 7A). In contrast, all of the glucosinolates derived frommethionine derivatives extended by five or six methyleneresidues were still present. These results suggest thatCYP79F1 specifically catalyzes the oxidation of short-chainmethionine derivatives and does not use the long-chain de-rivatives as substrates (Figure 7B).

Interestingly, the bus1-1f mutant contains higher concen-trations of glucosinolates derived from the long-chain me-thionine derivatives than do wild-type plants (Figure 6). Thelack of conversion of short-chain methionine derivatives totheir aldoximes probably leads to a buildup of these inter-mediates, which can then undergo further rounds of chainelongation, resulting in the formation of more glucosinolateswith longer side chains (Figure 7A). An increase of the tryp-tophan-derived indole-3-ylmethyl-glucosinolate was alsofound in mutant leaves and seed but not in roots.

Auxin Analysis

The biosynthesis of indolyl glucosinolates starts with theconversion of tryptophan to indole-3-acetaldoxime (IAOx)(Halkier, 1999; Hull et al., 2000; Mikkelsen et al., 2000). Thisreaction also might be the first step in the biosynthesis ofthe auxin IAA via a tryptophan-dependent pathway involvingIAN as an intermediate (Trp→IAOx→IAN→IAA) (Slovin et al.,1999). If IAOx is an intermediate in both IAA and indole glu-cosinolate biosynthesis, the two pathways might share reg-ulatory features. The increased concentration of indole-3-ylmethyl-glucosinolate in leaves and seed of bus1-1f (Fig-ure 6) raised the possibility that the auxin concentration waschanged as well. The morphology of the bus mutant (bushyphenotype and crinkled leaves) also suggested that it was al-tered in auxin concentration, given the important role that auxinplays in apical dominance and leaf morphology (Fellner,1999; Procházka and Truksa, 1999). Therefore, we mea-sured the concentrations of free IAA and its precursor IAN inleaves of bus1-1f and wild-type plants. The concentration ofboth compounds was higher in the mutant than in the wildtype: IAA was increased twofold (14 6 3.5 compared with7.6 6 1.2 pg/mg dry weight; n 5 3), and IAN was increasedfivefold (7.7 6 2.3 compared with 1.7 6 0.3 ng/mg dryweight; n 5 3). Therefore, it is possible that the observed busphenotype is caused by an increased concentration of auxin.

Overexpression of CYP79F1 in Arabidopsis

The lack of aliphatic glucosinolates with short carbon chainlengths in the bus1-1f mutant indicated that CYP79F1 oxi-dizes short-chain methionine derivatives. To further test thishypothesis, we determined the glucosinolate content ofwild-type plants overexpressing CYP79F1. We postulatedthat if the expression of the CYP79F1 gene results in thesynthesis of rate-limiting amounts of enzyme catalyzing theN-hydroxylation of short-chain methionine derivatives, over-expression would increase the levels of the correspondingglucosinolates.

When the CYP79F1 cDNA was expressed under the con-trol of the constitutive cauliflower mosaic virus (CaMV) 35S

Figure 6. (continued).

(A) Extracts of roots grown in vitro, rosette leaves, and seed from wild-type plants (wt) and bus1-1f mutants were analyzed by HPLC (see Meth-ods). Concentrations are given in mmol/g dry weight for roots and rosette leaves and mmol/g fresh weight for seed (values shown are means 6SD;n 5 2 for roots, and n 5 5 for leaves and seed). (2), not detected, and (1), that the peak was not well resolved from adjacent peaks and couldnot be quantified in all measurements. 3MSOP, 3-methylsulfinylpropyl; 3BZO, 3-benzoyloxypropyl; 4MTB, 4-methylthiobutyl; 4MSOB, 4-methyl-sulfinylbutyl; 4OHB, 4-hydroxybutyl; 4BZO, 4-benzoyloxybutyl; 5MTP, 5-methylthiopentyl; 5MSOP, 5-methylsulfinylpentyl; 6MSOH, 6-methylsul-finylhexyl; 7MTH, 7-methylthioheptyl; 7MSOH, 7-methylsulfinylheptyl; 8MTO, 8-methylthiooctyl; 8MSOO, 8-methylsulfinyloctyl; I3M, indole-3-ylmethyl; 1MOI3M, 1-methoxyindole-3-ylmethyl; 4MOI3M, 4-methoxyindole-3-ylmethyl.(B) Representative HPLC chromatograms from leaf extracts of one wild-type plant (wt) and one bus1-1f mutant. Signals are given in arbitraryunits (mAU). Peaks that were absent in the bus1-1f mutant are marked by arrows. Abbreviations are as given in (A).

360 The Plant Cell

promoter in Arabidopsis wild-type plants, 90% of the trans-genic plants (32 of 35) exhibited a more or less pronouncedbus phenotype, indicating different degrees of silencing ofthe CYP79F1 gene. The remaining transgenic plants did notshow any morphological changes compared with wild-typeplants. RT-PCR analysis confirmed that the transcript levelof CYP79F1 was reduced in leaves of transgenic plants ex-hibiting the bus phenotype, whereas the transcript level wasincreased in leaves of transgenic plants exhibiting the wild-type phenotype (Figure 8A). By segregation and DNA gelblot analyses, we identified a homozygous, CYP79F1-over-expressing line carrying a single T-DNA insertion. Seed ofthis line had a higher content of short-chain methionine-derived glucosinolates compared with that of wild-typeseed. The aliphatic glucosinolates with the shortest carbonchain lengths were especially enriched (Figure 8B). For ex-ample, 3-methylthiopropyl and 3-methylsulfinylpropyl, whichwere not detected in the seed of wild-type plants, werepresent in the transgenic line. On the other hand, the con-centration of methionine-derived glucosinolates with longcarbon side chains was reduced in the overexpressing line

compared with the wild type. Overexpression of CYP79F1led to an enhanced conversion of short-chain methioninederivatives to their aldoximes and consequently to a higherconcentration of glucosinolates with short carbon chain lengths(the only exception was a decrease of 4-benzoyloxybutyl).The more rapid use of short-chain methionine derivativesprobably leaves less substrate for further chain elongationand thus results in lower concentrations of aliphatic glucosi-nolates with long side chains.

DISCUSSION

Cytochrome P450s and Glucosinolate Biosynthesis

Glucosinolate biosynthesis is initiated by the oxidation ofamino acids to their corresponding aldoximes. Several dif-ferent types of enzymes have been implicated in this con-version: plasma membrane–bound peroxidases in theformation of indolyl glucosinolates from tryptophan in Chi-

Figure 7. Proposed Model for the Function of CYP79F1 and CYP79F2.

(A) Scheme of the biosynthetic pathway for methionine-derived glucosinolates. Glucosinolates that are absent in the bus1-1f mutant are boxed.The lack of these glucosinolates is most likely caused by the blocked conversion (interrupted arrows) of short-chain methionine derivatives totheir aldoximes due to the absence of CYP79F1. Long-chain methionine derivatives might be converted by CYP79F2, as indicated by the ques-tion marks. Glucosinolate abbreviations are defined in the legend to Figure 6.(B) CYP79F1 is proposed to react exclusively with homo- to tetrahomo-methionine (n 5 3 to 6).

bus, a CYP79F1 Knockout Mutant 361

nese cabbage (Ludwig-Müller and Hilgenberg, 1988), flavin-containing monooxygenases in the formation of aromaticand aliphatic glucosinolates from homophenylalanine andmethionine derivatives (Bennett et al., 1993, 1996), and cy-tochrome P450s in the formation of aromatic glucosinolatesfrom phenylalanine and tyrosine in white mustard, nastur-tium, and papaya (Du et al., 1995; Du and Halkier, 1996;Halkier and Du, 1997). This study unambiguously demon-strated the involvement of the cytochrome P450 CYP79F1in the formation of aliphatic glucosinolates from chain-elon-gated methionine derivatives. The lack of short-chain ali-phatic glucosinolates in the null mutant and the increasedlevels in the overexpressing plant indicated that CYP79F1uses short-chain methionine derivatives as substrates. Thisis further supported by heterologous expression studies ofCYP79F1 in E. coli, which revealed that recombinantCYP79F1 is able to convert dihomo- and trihomomethionineto their corresponding aldoximes (Hansen et al., 2000). To-gether with the recent cloning of CYP79B2 and CYP79B3involved in indolyl (Hull et al., 2000; Mikkelsen et al., 2000)and CYP79A2 involved in aromatic (Wittstock and Halkier,2000) glucosinolate biosynthesis, our results suggest thatin Arabidopsis the cytochrome P450s of the family CYP79perform the initial aldoxime formation for all types of gluco-sinolates.

CYP79F1 Expression Pattern and Substrate Specificity

The expression pattern of CYP79F1 provides valuable infor-mation regarding the site of glucosinolate biosynthesis inArabidopsis. Glucosinolates in this species accumulate in alltissues that have been analyzed so far, including roots,leaves, and seed (Haughn et al., 1991) (Figure 6). Inspectionof the glucosinolate content of various organs revealed amuch higher concentration of short-chain methionine-derivedglucosinolates in leaves than in roots. This difference corre-lates well with the higher expression level of CYP79F1 wefound in leaves as opposed to roots (Figure 5D).

In other species (white mustard and rapeseed), it hasbeen demonstrated that one of the major sites of glucosino-late biosynthesis is the silique wall (Toroser et al., 1995a,1995b; Du and Halkier, 1998). Glucosinolates are trans-ferred from the walls to the seed, where they accumulateduring seed maturation. The expression of CYP79F1 in thevascular strands of silique walls and seed funicles suggestsa similar mode of glucosinolate synthesis and deposition inArabidopsis.

The involvement of CYP79F1 in the biosynthesis of ali-phatic glucosinolates was clearly shown by the completeabsence of short-chain methionine-derived glucosinolatesin the null mutant and their increased concentration inCYP79F1-overexpressing plants. However, the metabolicfunction of the neighboring gene, CYP79F2, with 89% iden-tity to CYP79F1 at the amino acid level, is still uncertain.CYP79F2 does not appear to catalyze the same reactions

as CYP79F1 because it fails to assume the function ofCYP79F1 in the null mutant bus1-1f, although it is ex-pressed in the same cells of apical plant parts. It might bepostulated that CYP79F2 converts long-chain methioninederivatives to their corresponding aldoximes (Figure 7). Thishypothesis would explain the striking differences in the ex-pression levels of CYP79F1 and CYP79F2 in roots: the lowexpression of CYP79F1 coincides with a low content ofshort-chain methionine-derived glucosinolates in these or-gans, and the high expression of CYP79F2 coincides with ahigh content of long-chain methionine-derived glucosino-lates (Figure 6). To study this problem in more detail, thepreparation of knockout mutants for CYP79F2 is currentlyunder way.

The bus Phenotype

The bus phenotype may result from either an altered contentof methionine-derived glucosinolates and their biosyntheticintermediates or from an increase of indolyl glucosinolatesand IAA concentrations. There are several lines of evidencethat suggest a role for auxin in the production of the busphenotype. First, the changes in indolyl glucosinolate con-centrations observed could result in changes in IAA concen-trations, given the postulated metabolic links between thesetwo groups of substances (Ludwig-Müller and Hilgenberg,1988). Second, the intimate relations between auxin andbranching, vascularization, and leaf shape, reported in theliterature for many years, suggest the involvement of auxinin any mutant with abnormalities in these traits (Davies,1995; Fellner, 1999; Procházka and Truksa, 1999). Althoughenhanced shoot branching is traditionally correlated withdecreased auxin concentrations, the bus1-1f mutant actu-ally contained increased auxin levels. However, during thelast decade, it became clear that changes in hormone con-centrations in mutant plants often do not correlate well withthe degree of branching (Napoli et al., 1999). For example,the pea mutant rms-2 that displays a highly branched phe-notype has an increased instead of a decreased auxin level(Beveridge et al., 1994).

Alternately, the modified profile of chain-elongated me-thionine derivatives in the bus1-1f mutant may result inchanges in methionine metabolism that affect plant growthand development. Besides its role as a structural compo-nent of proteins, methionine serves as the direct precursorof both S-adenosylmethionine, the principal methyl donor intrans-methylation reactions, and the hormone ethylene(Ravanel et al., 1998). In addition, methionine is an immedi-ate progenitor of S-methylmethionine, a major sulfur trans-port form (Bourgis et al., 1999). The genetic lesion inCYP79F1 in bus1-1f may lead to the buildup of chain-elon-gated analogs of methionine, which may in turn interferewith methionine synthesis, utilization, or transport, leadingto changes in gross morphology. Arabidopsis mutants thatare deficient in methionine (Ravanel et al., 1998; Kim and

362 The Plant Cell

Leustek, 2000) or that overaccumulate methionine (Bartlemet al., 2000) show reduced growth compared with wild-typeplants.

Glucosinolates in Crop Improvement and Food Design

The nutritional value of rapeseed meal as animal feed is re-duced by high glucosinolate content. For this reason, over40 years ago plant breeders initiated a search for rapeseedthat is low in glucosinolates. In 1968, seeds from plants ofthe Polish Brassica napus variety Bronowski were found tobe low in glucosinolates (Josefsson and Appelqvist, 1968).This genetic source was then incorporated into high-yieldingvarieties. However, the molecular basis of the low glucosi-nolate content in this variety is still poorly understood anddoes not appear to result from an obvious biosyntheticblock. The identification of genes encoding cytochromeP450s of the family CYP79 as key enzymes in the glucosi-nolate biosynthesis pathway provides additional tools tomodulate glucosinolate profiles.

Manipulation of glucosinolate composition could also im-prove the pest resistance of rapeseed, because glucosino-

lates influence the behavior of insect, avian, and molluscanherbivores and are powerful antibacterial and antifungalagents (Louda and Mole, 1991). For example, surveys of her-bivore damage on rapeseed lines with different glucosinolatecontents have shown that increased glucosinolate levels re-duce feeding by pigeons and slugs but increase feeding byspecialist insects such as flea beetles (Giamoustaris andMithen, 1995). However, compositional changes, such as in-creasing the length of the side chain of methionine-derivedaliphatic glucosinolates, decreased flea beetle damage.Hence, altering the chain length of aliphatic glucosinolates, aswas achieved in this study by producing a null mutant ofCYP79F1 in Arabidopsis, may have agricultural value.

Recent interest in glucosinolates also derives from theirrole as potential anticancer agents in human diets (Jongen,1996; Verhoeven et al., 1997). For example, 4MSOB isothio-cyanate, a hydrolysis product of 4MSOB found in broccoli,was demonstrated to be a potent anticarcinogen (Zhang etal., 1992). Hence, increasing the concentration of the short-chain methionine-derived glucosinolate 4MSOB, which wasachieved in this study by overexpressing CYP79F1 in Arabi-dopsis, may increase the anticancer properties of glucosino-late-containing foods. Additional experiments are necessary

Figure 8. Overexpression of CYP79F1 in Transgenic Arabidopsis Plants.

The CYP79F1 cDNA was expressed in Arabidopsis under the control of the CaMV 35S promoter. wt, wild type.(A) RT-PCR reactions using poly(A)1 RNA from rosette leaves. Transgenic plants exhibiting the bus phenotype (1) had lower transcription levelsof CYP79F1 than did wild-type plants, whereas transgenic plants without the bus phenotype (2) had higher transcription levels.(B) Glucosinolate pattern in seed of the homozygous plant 35S::CYP79F1 overexpressing CYP79F1 compared with the wild type. Concentra-tions are given in mmol/g (means 6SD; n 5 5). Glucosinolate abbreviations are defined in the legend to Figure 6.

bus, a CYP79F1 Knockout Mutant 363

to determine if this strategy can be applied successfully toother species.

METHODS

Plant Material and Growth Conditions

Arabidopsis thaliana ecotype Columbia was the wild type used forcrossing and transformation. The bus mutants have been identifiedin an En-1–mutagenized collection of Arabidopsis ecotype Columbia(Wisman et al., 1998).

Plants were grown in soil either in a greenhouse at 208C with a 16-hr light period or in a growth chamber at 228C with an 8-hr light pe-riod (short-day conditions). For in vitro culture, seed were surface-sterilized and plated on agar-solidified half-strength Murashige andSkoog (1962) (MS) medium (2.15 g/L MS salts, 1% sucrose, pH 5.8,with KOH). Plates were grown in a culture chamber using 16-hr lightperiods at 150 mE m22 sec21, 75% humidity, at 218C. For Arabidopsisroot liquid cultures, z20 sterilized seed were grown in 40 mL of MSmedium (4.3 g/L MS salts, 3% sucrose, pH 5.8, with KOH) in Erlen-meyer flasks and agitated at 90 rpm for 3 weeks. Tobacco BY2 cellswere grown according to Nagata et al. (1981) and subcultured weekly.

Segregation Analysis of Plant 4L9-1 and Isolation of Flanking Genomic DNA

The F1 progeny of plant 4L9-1 segregated 3:1 for the bus phenotype.The genotype of F1 plants exhibiting the wild-type phenotype (eitherheterozygous for the bus1-2 allele or wild type) was determined byphenotypical analysis of the F2 generation. Individual plants with de-fined genotypes were used in DNA gel blot experiments to determineif one of the four En-1 insertions cosegregates with the bus pheno-type. Isolation of genomic DNA and DNA gel blot analysis were per-formed as described (Sambrook et al., 1989). DNA was digested withCsp6I. Hybridization was performed using the 59 end of En-1 (z200bp) as a probe. Analysis revealed an 800-bp Csp6I fragment (600 bpof En-1 plus 200 bp of flanking genomic DNA) cosegregating with thephenotype. Flanking genomic DNA was isolated from a homozygousmutant plant by ligation-mediated polymerase chain reaction (PCR)(Varotto et al., 2000) and used as a probe on the same DNA gel blotdescribed above. Hybridizing Csp6I fragments of the wild type andthe mutant allele were different in length because of the Csp6I re-striction site in En-1. The results confirmed that plants homozygousfor the bus phenotype were homozygous for the insertion inCYP79F1, plants heterozygous for the bus phenotype were het-erozygous for the insertion, and plants with the wild-type phenotypedid not contain an insertion in CYP79F1. Cosegregation analysis re-vealed that none of the other En-1 insertions could be responsible forthe phenotype. The possibility that the phenotype was caused by astable footprint after En-1 excision from another closely linked genewas excluded based on the identification of plants that had revertedto wild type in offspring populations of homozygous bus1-2 mutants.

Genetic Analysis of bus1-1 and Identification of bus1-1f

The En-1 element in bus1-1 was identified in PCR reactions by usinggenomic DNA as a template (10 ng; for conditions, see Reverse Tran-

scription–PCR Analysis below) and the following primer combina-tions: the CYP79F1 forward primer 59-ATGATCATGTGATGTTTTGTC-ACAGGG-39 (genomic region 64,929 to 64,955 of bacterial artificialchromosome [BAC] clone F309) and the En-1 primer 59-GCTCCAATG-ACCCACCAACAGAATG-39 as well as the CYP79F1 reverse primer59-CCACAAACATAATCAAAGACTACTCTGT-39 (genomic region65,613 to 65,640) and the En-1 primer 59-AGAAGCACGACGGCT-GTAGAATAGGA-39.

The bus1-1 mutant was backcrossed to Arabidopsis wild-typeplants for two generations. Plants were allowed to self-propagate,and their progeny were tested for the segregation of the bus pheno-type. In PCR reactions with the primer combinations mentionedabove, the En-1 insertion could not be detected. Amplification withthe two CYP79F1-specific primers (see above) and subsequent se-quencing led to the identification of bus1-1f.

Reverse Transcription–PCR Analysis

Tissue-Specific Expression Studies

Poly(A)1-RNA was isolated from various tissues by using oligo(dT)25

Dynabeads and following the manufacturer’s instructions (Dynal,Oslo, Norway). Roots were harvested from liquid cultures grown invitro. All other tissues were harvested from plants grown in thegreenhouse. Fifty nanograms of poly(A)1 RNA was reverse tran-scribed with 200 units of Superscript II reverse transcriptase (LifeTechnologies, Grand Island, NY) in a 50-mL reaction containing 1 3first strand buffer (Life Technologies), 0.5 mg of oligo(dT)12-18 (Amer-sham, Uppsala, Sweden), 0.2 mmol of DTT, and 25 nmol of eachdeoxynucleotide triphosphate, according to the manufacturer’s in-structions (Life Technologies). cDNA samples (5 mL) were used forsubsequent PCR amplifications. The CYP79F1 cDNA fragment (1.6 kb)was amplified with the forward primer 59-TCCATGGCATCAATC-ACTCTACTGG-39 (genomic region 63,931 to 63,955 of BAC cloneF309) and the reverse primer 59-AAGCGTTGAAGAAGATAACAGA-39

(genomic region 66,179 to 66,200). The CYP79F2 cDNA fragment (0.9 kb)was amplified with the forward primer 59-TCAACTTTCCATCGTAGA-GTCCATT-39 (genomic region 60,584 to 60,608) and the reverse primer59-GGTATCTTGACGGGCAACATGAGGT-39 (genomic region 61,607 to61,631). The actin-2 cDNA fragment (1.1 kb) was amplified with the for-ward primer 59-TTCCTCAATCTCATCTTCTTCC-39 and the reverseprimer 59-GACCTGCCTCATCATACTCG-39. PCR was performed in 1 3buffer (Hoffmann-La Roche, Mannheim, Germany) containing 1.5 mMMgCl2, 0.05 mM each deoxynucleotide triphosphate, 1 unit of Taq DNApolymerase (Hoffmann-La Roche), and 20 pmol of each primer, for 35cycles at an annealing temperature of 588C.

Cloning of CYP79F1 and CYP79F2 cDNAs

Reverse transcription (RT)–PCR reactions with poly(A)1 RNA isolatedfrom rosette leaves were performed as described above. CYP79F1cDNA was amplified with the forward primer 59-ATACATCATGAT-GAGCTTTACC-39 (genomic region 63,864 to 63,885 of BAC clone F309)and the reverse primer 59-AAGCGTTGAAGAAGATAACAGA-39 (ge-nomic region 66,179 to 66,200), and CYP79F2 cDNA was amplifiedwith the forward primer 59-TCAAACAAAAATACAAACATTA-39 (ge-nomic region 60,206 to 60,227) and the reverse primer 59-ACAAAG-CGTCGAAACACATC-39 (genomic region 63,318 to 63,337). Theforward primers contained an additional XbaI restriction site and the

364 The Plant Cell

reverse primers an additional BamHI restriction site. CYP79F1 cDNAwas subcloned into pUC18, and CYP79F2 cDNA was subcloned intopBluescript SK2. Both cDNAs were sequenced by the DNA core fa-cility at the Max-Planck-Institut für Züchtungsforschung (Cologne,Germany) on PE-Applied Biosystems (Weiterstadt, Germany) AbiPrism 377 and 3700 sequencers by using BigDye terminator chemistry.

Expression of N-Terminal Green Fluorescent Protein Fusionsin BY2

The 59 region of CYP79F1 was isolated from the cloned cDNA (seeReverse Transcription–PCR Analysis) by RcaI digestion (the RcaI siteis just upstream of the start ATG). The resulting 180-bp fragment wassubcloned into the NcoI site of the vector pCATSgfp (kindly providedby Guido Jach, Max-Planck-Institut für Züchtungsforschung). It con-tains a codon-optimized green fluorescent protein (GFP) S65C mu-tant gene (Heim et al., 1995) under the control of the cauliflowermosaic virus (CaMV) 35S promoter and an additional translation-enhancing element of tobacco etch virus (the basic vector is de-scribed in Carrington and Freed, 1990). The 59 region of CYP79F2was isolated by PCR using genomic DNA as a template (10 ng; forconditions, see RT-PCR Analysis) and the following primer combina-tion: forward, 59-TTAAGATGATGATGAAGATT-39 (genomic region60,235 to 60,254 of BAC clone F309, with an additional EcoRI site atthe 59 end), and reverse, 59-AATTGTCTCCAATGGACTCT-39 (ge-nomic region 60,599 to 60,618). The PCR product (400 bp) was di-gested with EcoRI and RcaI, leading to a 180-bp fragment that wassubcloned into the vector pCATSgfp digested with EcoRI and NcoI,thereby replacing the translation-enhancing element. As a control forendoplasmic reticulum (ER) localization, the GFP variant mGFP5ER(Haselhoff et al., 1997), which has been cloned into the vector pBT4(Feldbrügge et al., 1994), was used. As a control for cytoplasmic lo-calization, the vector pCATSgfp (see above) was used. Transient ex-pression in tobacco BY2 protoplasts and microscopic analysis wereperformed as described by Bischoff et al. (2000).

Construction of Binary Vectors

PCYP79F1 and PCYP79F2 were isolated by PCR using genomicDNA as a template (10 ng; for conditions, see Reverse Transcrip-tion–PCR Analysis). PCYP79F1 was amplified with the forwardprimer 59-ACTGTTGTTTTTAGACTATCCA-39 (genomic region 62,359to 62,380 of BAC clone F309) and the reverse primer 59-GATGAT-GTATGTATGCGTGTAG-39 (genomic region 63,852 to 63,873; thestart ATG has been mutated to ATC corresponding to GAT in thecomplementary strand). PCYP79F2 was amplified with the forwardprimer 59-ATTATAAGTGACTGTCTCATCT-39 (genomic region 59,606to 59,627) and the reverse primer 59-ATCTTAATGTTTGTATTTTTGT-39

(genomic region 60,220 to 60,241; the primer ends just before thestart ATG). The forward primers also contained an XbaI restrictionsite, and the reverse primers contained a BamHI site. PCYP79F2 wascloned as an XbaI-BamHI fragment into pBluescript SK2 and sub-sequently as an SstI-BamHI fragment into the binary vectorpVKH35SGUSpA (Reintanz, 1997; see below). PCYP79F1 containsan internal BamHI site and therefore was cloned in several steps intothe binary vector. First, the 59 800-bp XbaI-BamHI fragment was sub-cloned into pBluescript SK2 and then cloned as an SstI-BamHI frag-ment into pVKH35SGUSpA. Subsequently, the 39 700-bp BamHIfragment was ligated directly into pVKH35SGUSpA. This binary

vector allows plant selection on hygromycin and contains the uidAgene under the control of the CaMV 35S promoter. This promoterwas excised by digestion with SacI and BamHI and replaced byPCYP79F1 and PCYP79F2, respectively.

The CYP79F1 cDNA was isolated from pUC18 (see Reverse Tran-scription–PCR Analysis) and cloned into the binary vectorpVKH35SpA1 (Reintanz, 1997). This binary vector allows plant selec-tion on hygromycin and contains a CaMV 35S–poly(A) cassette with apolylinker in between. For cloning, HindIII and XbaI restriction sites inthe polylinker were used. The vector was digested with HindIII, over-hangs were filled in with T4 DNA polymerase, and the vector was re-digested with XbaI. Subsequently, CYP79F1 cDNA was ligated as anXbaI-SmaI fragment.

Generation of Transgenic Arabidopsis Plants

Arabidopsis plants were transformed by dipping developing flowersinto Agrobacterium cultures that had been grown overnight (Cloughand Bent, 1998). Seed were collected and screened for hygromycinresistance (half-strength MS medium supplemented with 15 mg/mLhygromycin). After 2 weeks, 35 resistant seedlings (T1 generation)were transferred to the greenhouse. The T2 generation was screenedfor 3:1 (resistant:nonresistant) segregation. To identify homozygousplants, the seed of 10 resistant plants from two T2 plant lines werescreened for 100% resistance (T3 generation).

Histochemical Assay of b-Glucuronidase Expression

Intact seedlings grown in vitro or excised organs of mature plantswere vacuum infiltrated with b-glucuronidase (GUS) staining solution(100 mM Na2HPO4, pH 7, 0.1% Triton-X 100, 2 mM K3Fe[CN]6, 2 mMK4Fe[CN]6, and 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-b-glucuronicacid) two times for 10 min each. After incubation overnight at 378C,tissue was cleared by treatment with 70% ethanol and analyzed withthe Leica microscope DM R (Leica, Wetzlar, Germany).

Glucosinolate Analysis

Rosette leaves were harvested from 6-week-old plants grown undershort-day conditions (there were no differences in the sizes of theleaves between bus1-1f and wild-type plants; see Figure 1D). Foreach sample, all rosette leaves from one plant were collected; fivesamples were measured. Roots were isolated from liquid culturesgrown in vitro, each culture containing z20 plants (one culture persample; two samples were measured). Roots and leaves were lyo-philized before measurement. For seed analysis of either the bus1-1fmutant or the transgenic line 35S::CYP79F1, five samples were mea-sured (each 25 mg).

Samples were immersed in 4 mL of boiling water containing 100mL of 0.3 M Pb(OAc)2/0.3 M Ba(OAc)2 solution and 0.5 mmol of inter-nal standard (allyl glucosinolate) for 15 min. After 30 min of gentleshaking at room temperature, samples were centrifuged at 4000g for10 min, and the supernatant was loaded onto a small (100 mg) col-umn of DEAE Sephadex A-25. The column was rinsed with 67%(aqueous) methanol and water. After capping, 50 mL of sulfatase so-lution (Graser et al., 2000) was loaded and incubated overnight. Theresulting desulfoglucosinolates were eluted from the column with 0.8mL of 60% (aqueous) methanol and 0.8 mL of water and pooled.

bus, a CYP79F1 Knockout Mutant 365

Separation of desulfoglucosinolates was achieved by HPLC on aHewlett-Packard (Palo Alto, CA) HP 1100 series system with au-tosampler, column oven, and diode array detector. The procedureused a C18 fully end-capped reverse phase column (LiChrospherRP-18, 250 3 4.6 mm i.d., 5-mm particle size; Chrompack, Raritan,NJ) operated at 1 mL/min and 258C. The system was equipped witha C18 LiChrospher (75 3 4.6 mm i.d., 5-mm particle size; Chrompack)reverse phase guard column. The injection volume was 20 mL. Elu-tion was accomplished with a gradient (solvent A, water; solvent B,acetonitrile) of 1.5 to 5% solvent B (6 min), 5 to 7% solvent B (2 min),7 to 21% solvent B (10 min), 21 to 29% solvent B (5 min), and 29 to57% solvent B (14 min), followed by a cleaning cycle (57 to 93% sol-vent B for 2 min, 5 min of hold, 93 to 1.5% solvent B for 3 min, and 6min of hold). Eluting compounds were monitored at 229 nm, andpeaks were identified by matching retention times and UV spectrawith those of desulfoglucosinolate standards. Concentrations of glu-cosinolates were calculated in relation to the internal standard by ap-plying response factors established for single desulfoglucosinolates(P.D. Brown and J. Gershenzon, unpublished data).

Indole-3-Acetic Acid (IAA) and Indole-3-Acetonitrile(IAN) Analysis

Rosette leaves were harvested from 3-week-old plants grown undershort day conditions (there were no differences in the sizes of theleaves between mutant and wild-type plants). For each sample, allrosette leaves from four plants were pooled, frozen in liquid nitrogen,and homogenized; three samples representing 12 plants were ana-lyzed.

Between 10 and 50 mg per sample was incubated with 1 mL of ex-traction buffer (50 mM Na2HPO4, pH 7, and 0.02% diethyldithiocar-bamic acid) and internal standards (250 pg of 13C6IAA and 20 ng of13C1IAN per sample) for 6 hr at 48C. Samples were centrifuged for 10min at 14,000g. The supernatants were acidified with 1 M HCl to pH2.7, and 60 mg of Amberlite XAD-7 was added. After 1 hr of incuba-tion, supernatants were removed, XAD-7 resin was washed with 1%acetic acid and vacuum dried, and absorbed compounds wereeluted twice with 1.5 mL of dichloromethane. Eluates were com-bined, vacuum-dried, and methylated with diazomethane. Methyl-ated samples were dried, dissolved in 15 mL of heptane, and silylatedwith 5 mL of N,O-bis-(trimethylsilyl)-trifluoracetamide/0.1% trimethylchlorosilane for 20 min at 708C, and 1 mL of each sample was split-less injected into a gas chromatography–mass spectrometry system(Edlund et al., 1995).

The column used for analysis was a CP-SIL 24CB (30 m 3 0.25mm; Varian, Inc., Lexington, MA), and the injector temperature wasset to 2708C. After injection, the column temperature was held at608C for 2 min, increased at a rate of 208C/min to 2008C, and then in-creased again at a rate of 48C/min to 2708C. Column effluent was in-troduced into the ion source of a JEOL JMS SX102/102 massspectrometer. The ion source and a gas chromatography interfacewere held at 2708C, and ions were generated with 70 electron volts atan ionization current of 300 mA. The mass spectrometer operated inselected reaction monitoring mode with an acceleration voltage of210 kV and the ion multiplier set to 21.5 kV. The precursor ions wereselected by magnetic switching; the daughter ions were selected bysimultaneous switching of the magnetic and electrostatic fields. ForIAA analysis, the monitored reactions were between mass-to-chargeratio (m/z) 5 261.118 and m/z 5 202.105 (endogenous IAA) or be-tween m/z 5 267.137 and m/z 5 208.125 (internal standard). IAN

measurement was based on reactions monitored between m/z 5

228.108 and m/z 5 213.085 (endogenous IAN) or between m/z 5

229.112 and m/z 5 214.088 (internal standard) (Ilic et al., 1996). Peakintegration and additional data processing were performed usingJEOL XMass software.

ACKNOWLEDGMENTS

We thank Dr. O. Leyser for helpful comments on the manuscript, ourservice group ADIS (Automated DNA Isolation and Sequencing) forDNA sequencing, ZIGIA (Center for Functional Genomics in Arabi-dopsis) for the En lines and support, and members of ourlaboratory, particularly A. Szyroki, L. Vahlkamp, and Dr. P. Wolff, forcritically reading the manuscript. This work was funded by the Euro-pean Community BIOTECH program and the International Coop-eration Copernicus program. We also acknowledge support by theEuropean Community V Frame work program (Grant No. QLRT-1999-01209).

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DOI 10.1105/tpc.13.2.351 2001;13;351-367Plant Cell

Sandberg, Matthias Godde, Rainer Uhl and Klaus PalmeBirgit Reintanz, Michaela Lehnen, Michael Reichelt, Jonathan Gershenzon, Marius Kowalczyk, Goran

Aliphatic Glucosinolates Knockout Mutant with Abolished Synthesis of Short-ChainCYP79F1, a Bushy Arabidopsis bus

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