The Plant Cell, Vol. 11, 1911–1923, October 1999, www.plantcell.org © 1999 American Society of Plant Physiologists

A Defect in

b

-Oxidation Causes Abnormal Inflorescence Development in Arabidopsis

Todd A. Richmond

1

and Anthony B. Bleecker

2

Department of Botany, University of Wisconsin–Madison, Madison, Wisconsin 53706

The

abnormal inflorescence meristem1

(

aim1

) mutation affects inflorescence and floral development in Arabidopsis. Af-ter the transition to reproductive growth, the

aim1

inflorescence meristem becomes disorganized, producing abnormalfloral meristems and resulting in plants with severely reduced fertility. The derived amino acid sequence of AIM1 shows

extensive similarity to the cucumber multifunctional protein involved in

b

-oxidation of fatty acids, which possesses

L

-3-

hydroxyacyl-CoA hydrolyase,

L

-3-hydroxyacyl-dehydrogenase,

D

-3-hydroxyacyl-CoA epimerase, and

D

3

,

D

2

-enoyl-CoAisomerase activities. A defect in

b

-oxidation has been confirmed by demonstrating the resistance of the

aim1

mutant to2,4-diphenoxybutyric acid, which is converted to the herbicide 2,4-D by the

b

-oxidation pathway. In addition, the loss ofAIM1 alters the fatty acid composition of the mature adult plant.

INTRODUCTION

The

b

-oxidation pathway includes the only enzymes thatcompletely degrade fatty acids by the sequential removal oftwo carbon units, resulting in the formation of acetyl–coen-zyme A (CoA). The process of

b

-oxidation is common to alleukaryotic and prokaryotic organisms. There are two majorsystems of

b

-oxidation, mitochondrial and peroxisomal.Both systems are present in animals, whereas

b

-oxidation infungi and plants appears to take place almost entirely in gly-oxysomes and peroxisomes. Plants are able to degradefatty acids completely within their peroxisomes, whereasanimals require an additional mitochondrial

b

-oxidation sys-tem because the peroxisomal

b

-oxidation system seems tofunction only as a chain-shortening system (Tolbert, 1981;Mannaerts and Debeer, 1982; Gerhardt, 1986). The

b

-oxida-tion systems in animals have been well characterized bothmolecularly and biochemically (reviewed in Kunau et al.,1995; Eaton et al., 1996; Hashimoto, 1996; Mannaerts andvan Veldhoven, 1996).

The discovery of a peroxisomal system of

b

-oxidation wasfirst made in plants (Cooper and Beevers, 1969), and formany years,

b

-oxidation research in plants has focused pri-marily on fatty tissues, including the mobilization of storagereserves from fatty tissues such as castor bean endospermand the mesophyll of fat-storing cotyledons (Cooper andBeevers, 1969; ap Rees, 1980). Although the enzymes of the

plant peroxisomal

b

-oxidation systems have been well char-acterized biochemically (see Gerhardt, 1992), it is not knownwhether the

b

-oxidation system plays as important a role inplants as it does in animals. Loss of a single bifunctionalprotein acting in the

b

-oxidation pathway in humans cancause multiple disease symptoms and is eventually fatal(Watkins et al., 1989).

We do not know what role

b

-oxidation enzymes play inthe normal development of plants. The

abnormal inflores-cence meristem1

(

aim1

) mutation is a novel mutation in aplant

b

-oxidation enzyme that affects the mature plant. Wepresent here the cloning and characterization of the

AIM1

gene. We discuss the consequences of the absence of oneof the key enzymes in the

b

-oxidation pathway and the pos-sible role of

AIM1

in Arabidopsis development.

RESULTS

Isolation of the

aim1

Mutant

A set of transgenic lines constructed by Agrobacterium-mediated seed transformation of

Arabidopsis thaliana

eco-type Wassilewskija (Ws) (Feldmann, 1991) was screened forinflorescence meristem mutants. The

aim1

mutant was foundsegregating in a single F

2

family. The inheritance of the

aim1

mutation is recessive. For reasons described below, the

aim1

mutant has severely reduced fertility and is primarilymaintained as a heterozygote. The mutant was backcrossedmore than five times before detailed characterization wasinitiated.

1

Current address: Department of Plant Biology, Carnegie Institutionof Washington, Stanford, CA 94305.

2

To whom correspondence should be addressed. E-mail bleecker@facstaff.wisc.edu; fax 608-262-7509.

1912 The Plant Cell

Phenotypic Characterization of the

aim1

Mutant

The vegetative phase of development in the

aim1

mutant issimilar to that of the isogenic Ws ecotype when plants aregrown under long-day conditions (

.

16 hr of daylight). Theleaves of the vegetative rosette are often slightly smaller and

darker green in

aim1

plants, but the differences in phenotypeat this stage are insufficient to score

aim1

plants unambigu-ously in segregating populations. The difference in vegetativedevelopment between wild-type Ws (Figure 1A) and

aim1

(Figure 1B) plants can be enhanced by growing them undershort-day conditions (8 to 12 hr of daylight). Rosette diame-

Figure 1. Phenotypic Characterization of the aim1 Mutant.

(A) Wild-type Col rosette grown under short-day conditions (8 hr of light). Bar 5 1 in.(B) aim1 rosette in the Col background grown under short-day conditions (8 hr of light). Bar 5 1 in.(C) Close-up view of young developing aim1 inflorescence grown under long-day conditions (.16 hr of light).(D) Wild-type Ws inflorescence.(E) Mature inflorescence showing the most severe aim1-conferred phenotype.(F) Close-up of the axis of a secondary branch from an AIM1 antisense plant.(G) Wild-type Ws plant.(H) aim1 plant.(I) aim1 plant from same F2 population as the plant shown in (H), demonstrating the range of phenotypes seen in the mutant.

b

-Oxidation in Arabidopsis 1913

ter is then reduced two- to threefold, and the leaves oftenhave a twisted appearance in

aim1

homozygotes (Figure 1B).All leaves appear to be equally affected, regardless of age.

The

aim1

mutant undergoes the transition to reproductivedevelopment at the same time as the wild-type plant. Shootapical meristems at the transition from vegetative to repro-ductive development were harvested from plants that weregrown under short days (8 hr of light) initially and then trans-ferred to long days (16 hr of light) to induce flowering. Shootapices of

aim1

plants harvested before the transition toflowering as well as 4 days after the transition were indistin-guishable from wild-type vegetative meristems when exam-ined by scanning electron microscopy (data not shown).

It is after this point that the mutant phenotype becomesmost pronounced. The primary inflorescence meristem of thewild-type Ws plant produces two or three aerial secondary in-florescence branches subtended by cauline leaves, with eachseparated by elongated internodes, and then produces 30 to40 flowers. Each of the secondary meristems produces two orthree vegetative nodes before converting to a flower-produc-ing meristem. Secondary inflorescence branches appearingfrom the rosette, called basal secondary branches, develop inthe same way. The result is a highly branched set of flower-bearing stems that is typical of Arabidopsis (Figure 1G).

The

aim1

plant initiates the same basic pattern of inflores-cence development as the wild type (Figure 1D). However,the conversion from leaf-producing meristems to flower-producing meristems is severely affected in the

aim1

mutant(Figure 1C). Shoot apices were harvested from long-dayplants when the primary inflorescence shoot was 3 to 5 cmabove the basal rosette. At this time, wild-type apices dis-played a typical spiral array of developing flower buds atvarious stages of development (Figure 2A). By contrast, awide range of abnormalities was observed in inflorescencemeristems from

aim1

plants (Figures 2B to 2H). Inflores-cence branches show a range of phenotypes. In the mostsevere cases, the inflorescence meristem does not produceany recognizable floral structures but rather produces only asmall mass of undifferentiated tissue that subsequentlyceases to develop all together. More often, the inflorescencemeristems will produce a few to several floral structures inwhat appears to be a normal spiral pattern before terminat-ing (Figure 1E). These inflorescence meristems terminate ineither flowerlike structures or undifferentiated masses of tis-sue. Inflorescence meristems harvested from

aim1

plantsgrown under short-day conditions were more similar to wildtype (Figure 2I), although the flower buds produced by thesemeristems were clearly abnormal (Figure 2J).

The basal secondary branches of the

aim1

mutant aremuch the same as those of the primary ones, although manymore secondary branches are initiated in

aim1

mutants thanin the wild type. A single

aim1

plant may have 10 to 200higher order inflorescences in the Ws (Figures 1H to 1I) andColumbia (Col) backgrounds but fewer in the Landsberg

erecta

background. Multiple third-, fourth-, or fifth-orderbranches arise nested in the axils of the cauline leaves on

these secondary branches, often with little or no internodeelongation (Figure 1F). It could not be determined whetherthe final structures in this reiterated pattern are inflores-cence meristems that have deteriorated to the point atwhich they can no longer produce additional inflorescencesor are determinate floral meristems that have failed to de-velop any normal floral organs. Increased branching, suchas that observed with the

aim1

mutant, is often associatedwith reduced fertility or sterility (Hensel et al., 1994).

Defects in Flower Development Caused by the

aim1

Mutation

The

aim1

mutant does not produce any normal, fertile flow-ers like those of the wild type (Figure 2K). Figure 2 shows thewide variation in appearance of the flowers and flowerlikestructures in the

aim1

mutant. Under long-day conditions,there are often many floral organs missing from each flower,and there are often homeotic conversions (Figures 2O and2P). In general, flowers from

aim1

plants grown under short-day conditions are much more normal in appearance, al-though the plant itself is still basically sterile. If allowed togrow for extended periods of time (12 to 16 weeks), a smallamount of seed (50 to 100 seeds) can be collected from

aim1

plants. Although this seed is darker in color than isseed of the wild type and is variable in size and shape, it isviable. The production of small amounts of seed from

aim1

plants shows that there is not an absolute block in eithermale or female fertility.

Two broad classes of flower types can be distinguished.In the first broad class, which represents

z

60% of the floralstructures when the plants are grown under long-day condi-tions, sepal growth appears arrested, leaving the interior ofthe flower exposed (Figure 2J). In the other broad class, se-pal growth is mostly normal, but the interior of the flowershows arrested or delayed development (Figure 2N). Table 1shows the percentage of each type of organ found in

aim1

flowers of this class. There are often multiple stamens miss-ing (Figure 2M), or stamens are arrested at an early stage ofdevelopment (Figures 2J and 2N). Sepals and the gynoe-cium are the least affected in the mutant, whereas petalsand stamens are often missing or abnormal (Figure 2L), inagreement with the floral development model that pointsto carpels and sepals as the ground state (Haughn andSomerville, 1988; Meyerowitz et al., 1991). Approximately10% of this type of

aim1

flower has the normal complementof floral organs yet is still sterile. The primary defect appearsto be in pollen development, because few mature pollengrains were seen during flower dissection.

Molecular Cloning of the

AIM1

Gene

To ensure that the phenotype of the

aim1

mutant was the re-sult of the T-DNA inserting in a gene and not from another

1914 The Plant Cell

Figure 2. Examination of Later-Stage Inflorescence Meristems under Long-Day and Short-Day Conditions.

(A) Wild-type inflorescence meristem with floral buds ranging from stages 1 to 6 (stages following Smyth et al., 1990).(B) Long-day aim1 inflorescence. The inflorescence terminates in structures that are unlike any of the normal floral stages.(C) Long-day aim1 inflorescence. Although several floral buds have been initiated, the meristem is abnormal, unorganized, and more roundedthan that of the wild type.(D) Four floral buds produced by an aim1 inflorescence.(E) Terminally differentiated aim1 inflorescence. The flower in the foreground shows an abnormal, lobed sepal. The flower in the rear has a fila-mentous structure commonly seen in aim1 flowers.(F) Long-day aim1 inflorescence.(G) Close-up of a wild-type inflorescence meristem grown under long-day conditions.(H) Close-up of a long-day aim1 inflorescence meristem (M). The 2 denotes a stage 2 floral bud.(I) Wild-type inflorescence meristem grown under short-day conditions.(J) Short-day aim1 inflorescence meristem. Stamen primordia (white arrowheads) are indicative of stage 5 flowers, yet the sepals (black arrows)have not yet covered the floral meristem, a stage 4 characteristic.(K) Wild-type flower.(L) aim1 flower.(M) aim1 flower. The gynoecium is flattened and is V-shaped at the top (arrow).(N) Young aim1 flower showing delayed or arrested development.(O) Gynoecium from a long-day aim1 flower.(P) Terminal aim1 flower. There are filaments tipped with stigmatic tissue (white arrowheads), a deformed carpal, and stigmatic papillae visibleon the gynoecium (black arrow).Bars in (A) to (P) 5 100 mm.

b-Oxidation in Arabidopsis 1915

mutation caused by the transformation process, cosegrega-tion analysis was performed. Individual mutants from an F2

population were examined by DNA gel blot analysis, usingthe right border of the T-DNA as a probe. Twenty-four of 24individual mutants examined contained the T-DNA (data notshown), giving a ,1% chance that the T-DNA insertion as-sorts independently of the mutation. The gene adjacent tothe T-DNA insert contains 18 exons with an average size of120 bp and 17 introns with an average size of 152 bp. TheT-DNA insertion is located 194 bp 59 of the predicted startcodon. This gene encodes a protein of 721 amino acids,with a predicted size of 77.9 kD.

BLAST (Altschul et al., 1990) searches using sequencesfrom genomic subclones showed an identical match ofz500 bp to the 39 end of two overlapping Arabidopsis ex-pressed sequence tags (ESTs) placed in the dEST database(Höfte et al., 1993).

Verification That the Phenotype of the aim1 Mutant Is Due to the Disruption of the Gene Associated with theT-DNA Insertion

Three experiments were performed to confirm that loss ofexpression of the gene associated with the T-DNA insertwas responsible for the phenotype of the aim1 mutant. Afragment adjacent to the tag was used to probe total RNAisolated from wild-type and aim1 inflorescences. A 2.5-kbmessage could be found in wild-type plants but not in theaim1 mutant (Figure 3A). Second, wild-type genomic se-quences encompassing the gene were cloned into a vectorcontaining hygromycin resistance and used to transform aline that was heterozygous for aim1. A T3 population wasfound that was homozygous for both hygromycin and kana-mycin resistance and failed to segregate for the aim1 muta-tion, indicating that the wild-type genomic sequence can

complement the aim1 lesion. Finally, wild-type plants weretransformed with an antisense construct composed of thecoding sequence of the gene of interest fused in reverse ori-entation to the cauliflower mosaic virus 35S promoter.Twelve of 24 transgenic lines generated with this constructdisplayed phenotypes similar to the original phenotype ofthe aim1 mutant (Figure 1F). Taken together, these resultsprovide compelling evidence that the phenotype of the aim1mutant is due to the loss of expression of the gene associ-ated with the T-DNA insert. Consequently, we refer to thisgene as the AIM1 gene.

Mapping of AIM1 and the Multifunctional ProteinGene AtMFP2

The AIM1 gene was mapped to chromosome 4, position72.4, using recombinant inbred lines (Lister and Dean,1993). The AtMFP2 gene was mapped to the top of chromo-some 3, 4.9 centimorgans from marker g4119, using a seg-regating population of F5 families (Chang et al., 1993).

Figure 3. RNA Gel Blot Analysis of the AIM1 Gene.

(A) RNA gel blot of total RNA (5 mg) from wild-type (Ws) and aim1 in-florescences, probed with DNA flanking the T-DNA insert.(B) Time course of AIM1 expression in developing seedlings. rRNArepresents the 45S rRNA precursor used as a control for equal load-ing. Lanes are labeled by age (in days) of tissue. Cont., continuous;L, leaf.(C) RNA gel blot analysis of AIM1 RNA levels, using total RNA (5 mg).R, root; L, rosette leaf; S, stem; C, cauline leaf; Si, silique; F, flower.(D) Comparison of AIM1 and AtMFP2 RNA levels in various maturetissues. Lanes are labeled as given above.

Table 1. Floral Organ Composition of aim1 Flowersa

Organb

No. of Organs Sepals Petals Stamens Gynoecium

0 0.3 6.3 0.8 1.51 4.8 7.8 3.0 96.02 11.5 16.3 9.3 1.53 18.3 22.3 14.0 1.04 65.3 47.5 24.55 34.86 13.8

a Only aim1 flowers that were externally normal in appearance wereexamined; n 5 400.b Values are given as percentages.

1916 The Plant Cell

The Predicted AIM1 Protein Is Similar to MFPs Involved in b-Oxidation

A comparison of the predicted AIM1 amino acid sequencewith sequences in the current protein databases (NCBI;BLAST network server) shows that it exhibits significantsimilarity to the cucumber multifunctional protein (CuMFP)(accession number CAA55630) acting in glyoxysomal fattyacid b-oxidation. CuMFP is a 79.0-kD plant glyoxysomalprotein that has been shown to possess L-3-hydroxyacyl-CoA hydrolyase (enoyl-CoA hydratase; EC 4.2.1.17),L-3-hydroxyacyl-dehydrogenase (HDH; EC 1.1.1.211),D-3-hydroxyacyl-CoA epimerase, and D3, D2-enoyl-CoAisomerase (EC 5.3.38) activities (Preisig-Müller et al.,1994). The predicted AIM1 gene product is 56% identicalto CuMFP across the entire length of the protein. In addi-tion, searches of the EST database using the predictedprotein sequence revealed a second MFP gene in Arabi-dopsis. Full-length cDNAs were isolated and sequenced forthis gene, which we have designated as AtMFP2. AtMFP2is more closely related to CuMFP than to AIM1, showing76% identity. AIM1 is as similar to AtMFP2 to as it is toCuMFP (57% identity). A number of other proteins in thedatabase also show similarity to AIM1. These proteins alsofunction in the b-oxidation pathway in a wide variety of or-ganisms, including humans, rats, fungi, and bacteria.

Based on enzymatic evidence from purified CuMFP (Preisig-Müller et al., 1994), the AIM1 gene product is expected tohave an isomerase domain in the first 125 amino acids of theprotein, a hydratase domain between amino acids 100 and200, and a dehydrogenase domain between amino acids390 and 590 (Figure 4A). An epimerase activity has been lo-calized to the N-terminal end of CuMFP, but it has not beendelimited explicitly.

Figure 4B shows an alignment of the region surroundingthe isomerase/hydratase PROSITE (Bucher and Bairoch,1994) consensus sequence of the AIM1 gene product withproteins known to have hydratase activity, includingCuMFP, the human peroxisomal bifunctional enzyme, andthe large a subunit of the Escherichia coli fatty acid oxi-dation complex (FADB). The AIM1 protein differs fromthe PROSITE consensus sequence for the isomerase/hydratase superfamily at only one amino acid residue.However, the nonconserved alanine in AIM1 (A123) indi-cated in Figure 4B is shared by all of the putative plantMFPs (data not shown), suggesting that plant MFPs mayhave a different isomerase/hydratase consensus than thePROSITE pattern.

Figure 4C provides an alignment of the region surroundingthe dehydrogenase PROSITE consensus sequence. TheAIM1 gene product differs from the PROSITE consensus se-quence for HDH in three places, but at each residue, CuMFPhas the same nonconserved change. Because CuMFP hasbeen shown to have HDH activity, it is likely that AIM1 hasHDH activity as well, although this has not been demon-strated.

Enoyl-CoA Hydratase Activity of the AIM1 Gene Product

To verify that AIM1 and AtMFP2 have the enzymatic activi-ties suggested by their amino acid sequences, both wereexpressed as fusions to glutathione S-transferase in a bac-terial overexpression system (rAIM1 and rAtMFP2). Enoyl-

Figure 4. Amino Acid Sequence Comparison of AIM1 with Otherb-Oxidation Enzymes.

For alignments, the PROSITE consensus pattern is underlined, andthe asterisks indicate residues where AIM1 does not match thePROSITE consensus. The residues shaded in black are those thatare identical in all proteins.(A) Domain structure of various multifunctional b-oxidation proteins(from Preisig-Müller et al., 1994). Boxes represent known domains. Pro-tein sequences are as follows, with accession numbers given in paren-theses: CuMFP, cucumber multifunctional protein (CAA55630);EcFADB, E. coli FADB protein (CAB40809); HuPBE, human peroxi-somal bifunctional enzyme (Q08426).(B) Alignment of the PROSITE consensus region (PDOC00150) ofthe isomerase/hydratase domain of the AIM1 protein. The symbol xis used for a position at which any amino acid is accepted. Ambigu-ities are indicated by listing the acceptable amino acids for a givenposition between square brackets. Repetition of an element withinthe pattern is indicated by following that element with a numericalvalue within parentheses. Proteins are as given above, with the addi-tion of AtMFP2, an Arabidopsis AIM1 homolog.(C) Alignment of the PROSITE consensus region (PDOC00065) ofthe dehydrogenase domain of the AIM1 protein.

b-Oxidation in Arabidopsis 1917

CoA hydratase activity was found in both proteins. The Km

values for both rAIM1 (115 mM) and rAtMFP2 (240 mM) aresimilar to those reported for the CuMFPs. It appears thatAIM1 has a higher affinity for short-chain acyl-CoAs thandoes AtMFP2, although definitive proof would require purifi-cation of the enzymes from plant tissue. These data confirmthat both AIM1 and AtMFP2 have an enzymatic activity as-sociated with b-oxidation of fatty acids.

Fatty Acid Composition

Lack of a functioning b-oxidation system may result in anaccumulation of long-chain fatty acids (LCFAs). Table 2shows the fatty acid composition of total lipids from maturewild-type and aim1 rosette leaves. As expected, there arechanges in the fatty acid composition. In particular, thereare changes in unsaturated 18-carbon fatty acids, includingsignificantly elevated levels of C18:2 and C18:1.

Germination Rate in the aim1 Mutant

b-Oxidation is known to play an important role in mobilizingseed lipid reserves in germinating seedlings. To test whetherthe aim1 mutation affects seed germination in any way,seedlings were grown on half-strength Murashige and Skoog(MS) plates (Murashige and Skoog, 1962) with no additionalcarbon source. As a recessive mutation, one-quarter of theseed from the heterozygous parent would be expected tofail to germinate or show poor seedling growth. Seed fromheterozygous plants was plated with wild-type controls onhalf-strength MS plates with and without 2% sucrose, inboth the light and the dark. No difference was seen in seedgermination or seedling growth under any conditions tested.

However, when the rare seeds from aim1 homozygousplants were tested, a different result was found. When

plated on half-strength MS media without sucrose, none ofthe seeds germinated (,0.1%). However when plated onhalf-strength MS media plus 2% sucrose, 12% of the seedgerminated. Because aim1 homozygotes from an aim1 het-erozygous parent show no dependence on sucrose for ger-mination, a maternal effect of the aim1 mutation on seedgermination is indicated.

The aim1 Mutant Is Resistant to the Herbicide2,4-Diphenoxybutyric Acid

A diminished capacity for b-oxidation in aim1 seedlings wasdemonstrated using the herbicide 2,4-diphenoxybutyric acid(2,4-DB). In the presence of a functioning b-oxidation sys-tem, nontoxic 2,4-DB is converted to toxic 2,4-D (Synerholmand Zimmerman, 1947; Garraway, 1970; Hayashi et al.,1998). Seeds from an aim1 heterozygote were grown verti-cally on plates using a range of concentrations from 0.1 to100 mM 2,4-DB. aim1 seedlings are resistant to 2,4-DB,which is consistent with a defect in b-oxidation. Table 3 showsthat there is a small window of concentrations at which aim1exhibited resistance to 2,4-DB. At concentrations .4 mM,root growth was completely inhibited in both wild-typeseedlings and the aim1 segregating population. At 2 mM2,4-DB, however, one-quarter of the population segregatingfor aim1 showed elongated roots, and when transplanted tosoil, all of the seedlings with the elongated roots had theaim1 phenotype. This result demonstrates that b-oxidationin vegetative tissues is diminished in the aim1 mutant.

Expression Pattern of the AIM1 Gene

The expression of the AIM1 gene in mature plants was ex-amined by both RNA gel blot analysis and by using anAIM1–b-glucuronidase (GUS) reporter construct. RNA wasisolated from various tissues of mature Arabidopsis plantsand probed using the full-length AIM1 cDNA. The AIM1 tran-script proved to be expressed at approximately equal levelsin all tissues examined (Figure 3C). Only a single transcriptwas detected, in accordance with DNA gel blot data indicat-ing that AIM1 is a single-copy gene (data not shown). Ex-pression of AtMFP2 was examined in roots, rosette leaves,flowers, and siliques and compared with that of AIM1. Ineach tissue examined, AIM1 and AtMFP2 were expressed atapproximately equal levels (Figure 3D). Expression of AIM1was examined in developing seedlings grown in both thelight and the dark. Only faint expression was seen in eti-olated seedlings during days 3 through 6. Strong expressionwas detected only in older seedlings (.8 days) grown incontinuous light (Figure 3B). However, full-length cDNAswere isolated from a cDNA library made from 3-day-olddark-grown seedlings, indicating that both AIM1 andAtMFP2 are expressed at this stage of development, al-though expression may be low.

Table 2. Fatty Acid Composition of Leaves from the Wild Typeand aim1a

Percentage of Total

Fatty Acid Wild Type aim1

C16:0 0.20 6 0.01 0.18 6 0.01b

C16:1/16:2 0.06 6 0.01 0.07 6 0.01C16:3 0.13 6 0.01 0.12 6 0.01C18:0 0.01 6 0.01 0.01 6 0.00C18:1 0.01 6 0.01 0.03 6 0.00C18:2 0.08 6 0.01 0.14 6 0.01C18:3 0.51 6 0.02 0.46 6 0.01

a Fully expanded rosette leaves; n 5 5.b Numbers in boldface are significantly different from wild-type val-ues (P , 0.05).

1918 The Plant Cell

To examine the pattern of AIM1 expression in vegetative,inflorescence, and floral meristems, the putative AIM1 pro-moter region was placed upstream of the GUS reportergene in the Agrobacterium binary vector pBI101.2 (Jefferson,1987) to create a chimeric AIM1–GUS gene. Multiple trans-formants were obtained and three lines were chosen for fur-ther characterization because of the range of staining thatthey exhibited. All show the same pattern of expression butdiffer in the time it takes for GUS staining to develop fully.AIM1–GUS seedlings show GUS staining within the first 24hr after germination. There is high expression in the cotyle-dons and at the hypocotyl–root junction (Figures 5A and5B). The lack of staining in the midsection of the hypocotylmay be due to problems with penetration of the substrate asopposed to lack of expression, because other seedlingsshow darker staining in the hypocotyl. There is faint stainingin the roots. The expression pattern was evaluated in bothlight- and dark-grown seedlings and was shown to be simi-lar (data not shown). A rosette leaf (Figure 5E), stem, caulineleaf, inflorescence meristem, and flowers (Figure 5F) from amature plant all stain deeply, indicating strong expression inthese tissues as well. Roots from mature plants stain faintly,consistent with a lower level of expression detected by RNAgel blots.

Two-micron sections of GUS-stained tissue were exam-ined using dark-field optics, with GUS expression evident aspink crystals. Examination of 3-day-old seedlings showsthat the AIM1 promoter directs GUS protein expression in allcell types of the developing seedling (Figure 5B). Developingsiliques and embryos also show expression (Figures 5C and5D). There is staining in the interior of the silique near the de-veloping embryos, although the mature embryos show noevidence of GUS activity.

GUS staining is observed in the inflorescence meristemand in the earliest floral buds as well as in the mature flow-ers (Figures 5F and 5G). Stained inflorescences were sec-tioned and expression was seen in all cell types in themeristem and flowers, with particularly intense staining inthe developing anthers (Figure 5G). To confirm the GUS re-porter gene data, in situ hybridization was used to examineAIM1 gene expression in the inflorescence meristem. Figure5H shows expression of AIM1 throughout the inflorescencemeristem and in the developing flower buds, consistent withthe GUS data. No expression was detected when a sensecontrol probe was used.

DISCUSSION

Developmental Context of the Phenotype of theaim1 Mutant

The AIM1 gene encodes an MFP involved in the b-oxidationof fatty acids. AIM1 appears to be expressed in most tis-sues, and phenotypic effects of loss of function of this geneare detectable in seedlings and in the vegetative rosette.However, these effects on vegetative development are rela-tively minor. It is after the transition to reproductive develop-ment that the lack of AIM1 function has debilitating effectson growth and development.

We found a wide range of morphological phenotypes as-sociated with reproductive development in the aim1 mutant.Many of these phenotypes overlap with or are reminiscent ofphenotypes produced by other known mutations that affectreproductive development in Arabidopsis. Mutations in floralorgan identity genes may result in lack of development of or-gans within specific whorls of the flower and in homeoticconversions of organ identity in specific whorls (Weigel andMeyerowitz, 1994; Weigel and Clark, 1996). However, indi-vidual genes in this class affect primarily two adjacentwhorls in the flower, whereas the mutation in aim1 may af-fect any of the four whorls. Homeotic conversions were ob-served in aim1, but these were generally restricted tostructures arising directly from the inflorescence meristem.When organs were produced in floral meristems of aim1, theorgan identity was appropriate to the whorl. The chimericstructures observed at shoot apices of aim1 may be a pleio-tropic effect of floral programs invading the inflorescencemeristem. Such structures are commonly observed at theinflorescence apices of sterile plants (Hensel et al., 1994).

Biochemical Context of the aim1 Mutation

The AIM1 protein exhibits extensive similarity to enzymesresponsible for conducting the process of fatty acid b-oxi-dation. We have established that AIM1 has enoyl-CoA

Table 3. Growth of Seedlings on 2,4-DBa

Concentration of2,4-DB (mM) Population Elongated Roots/Short Roots

0 Ws 49:0aim1 F2

b 91:0

1 Ws 79:2aim1 F2 100:4

2 Ws 0:89aim1 F2 18:86c

4 Ws 0:100aim1 F2 0:103

8 Ws 0:96aim1 F2 0:108

a Representative trial; n 5 4.b aim1 segregating in the Ws background.c Not significantly different from a 1:3 segregation ratio at the P 5

0.05 level.

b-Oxidation in Arabidopsis 1919

Figure 5. Histochemical Localization of GUS Expression in Plants Transformed with AIM1–GUS.

(A) Seven-day-old seedling grown in continuous light.(B) Two-micron section from a 3-day-old seedling grown in continuous light. Staining, represented by the red crystals, is evident in all cell layers.(C) Developing silique from a 4-week-old plant.(D) Close-up of a 2-mm section from a cross-section of a mature silique.(E) Rosette leaf from a 4-week-old plant grown under 16-hr days.(F) Inflorescence from a 4-week-old plant grown under 16-hr days.(G) Close-up of a 2-mm section from a developing inflorescence.(H) In situ hybridization on mature inflorescence from a 4-week-old wild-type plant. There was no expression seen with a sense control probe(data not shown).

1920 The Plant Cell

hydratase activity, characteristic of an enzyme involved inb-oxidation. In addition, the inability of the aim1 mutant toproperly metabolize 2,4-DB to 2,4-D supports the theory thatAIM1 plays a role in the normal fatty acid metabolism of theplant via the b-oxidation pathway. This theory is supportedby the altered fatty acid composition of the aim1 mutant.

A total of four MFPs have been purified and characterizedfrom cucumber (Behrends et al., 1988; Gühnemann-Schäferand Kindl, 1995a, 1995b), although only one has beencloned and sequenced (Preisig-Müller et al., 1994). Three ofthese proteins have been isolated from cotyledons (MFPI toMFPIII), whereas the fourth (MFPIV) was isolated from leaftissue. The enzymatic activities of these four proteins differ;MFPI and MFPIV have no isomerase activity. In addition, thehydratase activity of these four proteins shows chain-lengthspecificity; MFP II shows no activity toward C18:1 fatty ac-ids, whereas MFPIV shows a higher activity toward shorterchain fatty acids (Gühnemann-Schäfer and Kindl, 1995a).Our work supports the presence of at least two MFPs inArabidopsis; AtMFP2 is more closely related to CuMFP(Preisig-Müller et al., 1994) than is AIM1.

In plants, b-oxidation of fatty acids provides energy and/or substrates for gluconeogenesis. Gluconeogenic b-oxida-tion is particularly important during seed germination, inwhich lipid reserves must be mobilized by conversion tosugars. Indeed, there are reported examples of deficienciesin b-oxidation that affect seed germination. Hayashi et al.(1998) devised a selection scheme for b-oxidation mutantsusing resistance to the herbicide 2,4-DB. Four of the 12 mu-tants they isolated show sucrose-dependent germination.This emphasizes the importance of b-oxidation in mobilizingseed lipid reserves in germinating seedlings. The lack of anadult phenotype in any of these mutants suggests that theremay be separate systems for mobilizing lipid reserves andfor normal housekeeping function and/or senescence-asso-ciated remobilization of lipids from leaves.

In contrast, the loss of AIM1 does not result in a sucrose-dependent germination phenotype. This may be due tofunctional redundancy among the MFPs in the developingseedling. It may be that there are other enzymes primarilyresponsible for mobilizing seed lipid reserves in the youngseedling, whereas AIM1 is responsible for conducting b-oxi-dation in nonfatty tissues. However, the activity of AIM1 isnot superfluous at this early developmental stage becauseaim1 seeds show reduced sensitivity to 2,4-DB; it is possi-ble that a higher affinity of AIM1 for short-chain fatty acidsas compared with AtMFP2 allows a small window of resis-tance for aim1 seedlings.

Reconciling the Developmental Phenotype of aim1 with the Biochemical Defect in b-Oxidation

We can consider three different hypotheses that account forthe developmental phenotype of the aim1 mutant. (1) b-Oxi-dation provides energy and reducing power from lipid re-

serves that are specifically needed during reproductivedevelopment. (2) b-Oxidation is used to produce a lipid me-tabolite(s) that plays a structural or signaling role in repro-ductive development. (3) The specific block in b-oxidation inaim1 results in the buildup of a lipid metabolite that disruptsintercellular communication in the reproductive meristems.

The first hypothesis is consistent with the notion that re-productive development has a higher energy demand asso-ciated with it than vegetative development. A precedent forthis idea is provided by forms of cytoplasmic male sterility inmaize (Levings, 1993). In the cms line, a specific defect inmitochondrial function in all tissues of the plant has a no-ticeable effect only on pollen development, ostensibly be-cause of the higher energy demand for male gametophytedevelopment (Levings, 1993). Although AIM1 is expressed inthe reproductive meristems, it is unlikely to function cell au-tonomously in energy generation in meristems because nolipid reserves are present in these tissues. On the otherhand, senescence of rosette leaves occurs concomitantlywith the transition to reproductive development (Hensel etal., 1994); thus, failure to convert lipids stored in leaf tissuesduring senescence could affect the energy budget of theplant during this phase of development. However, observa-tion of vegetative growth is inconsistent with this hypothe-sis. Vegetative development, particularly of stem andcauline leaf tissue, is quite vigorous in the mutant. In fact, at12 weeks after germination, the aim1 meristems producedmore total biomass than did their wild-type counterparts(data not shown).

The hypothesis that aim1 is blocked in the production of alipid signaling molecule is intriguing. However, so little infor-mation is available as to how intercellular signaling is usedto set up morphogenic fields in plant meristems that themerits of this hypothesis are difficult to evaluate. Most iden-tified genes that affect pattern formation in reproductivemeristems are presumptive transcription factors that arelikely to act cell autonomously (Weigel and Meyerowitz,1994; Weigel and Clark, 1996). However, the recent discov-ery that the terminal flower gene is related to lipid bindingproteins is consistent with the possibility that lipid signalingmay play a role in patterning associated with plant morpho-genesis (Bradley et al., 1997). In this regard, there are anumber of fatty acid–derived signals in plants: jasmonicacid, the traumatin family and related alkenals (Farmer,1994), highly oxygenated fatty acid derivatives reminiscentof animal lipoxins (Farmer, 1994), and lipooligosaccharides(Spaink et al., 1991). Fatty acids also have been shown tomodulate protein kinases in plants (Scherer, 1996, and refer-ences therein), which could provide a connection betweenaim1 and the phenotypically similar tousled (tsl ) mutant thatresults from loss of function of a protein kinase (Roe et al.,1993).

Perhaps the most plausible hypothesis to account for thephenotypic effects of aim1 is that a block in b-oxidationleads to the accumulation of intermediate or unique lipidmetabolites. These might disrupt the process of reproduc-

b-Oxidation in Arabidopsis 1921

tive development in a variety of ways. In humans, peroxiso-mal bifunctional enzyme deficiency results in multiplebiochemical abnormalities, including elevated levels of verylong chain fatty acids (VLCFAs) in both plasma and fibro-blasts, impaired b-oxidation in cultured fibroblasts, andabnormal bile acid metabolism (Watkins et al., 1989). Ac-cumulation of VLCFAs can have dramatic effects on plantmorphology (Millar et al., 1998). Although there is no accu-mulation of VLCFAs in the aim1 mutant, there are significantchanges in LCFAs that warrant further investigation.

Concluding Statement

There is much work to be done with aim1 to determine thelink between b-oxidation and normal plant development.There are at least two MFPs involved in b-oxidation in Arabi-dopsis. Loss of function of one of these genes results inabnormal inflorescence development. The aim1 mutation af-fects normal patterning at the inflorescence meristem andthe maintenance of cell growth, as is apparent by the de-layed development seen when examining flowers. Althoughthe fatty acid composition of the mutant is altered, there isno clear indication of how this affects plant development.The link between b-oxidation and development has beenestablished but not elucidated. However, this link holds thepromise of establishing a new role for lipid metabolism inregulating plant growth and may lead to the discovery ofnew lipid signaling molecules.

METHODS

Growth of Plants

Plants (Arabidopsis thaliana) were grown as previously described(Hensel and Bleecker, 1992; Hensel et al., 1993). Seeds were surfacesterilized with 30% bleach/0.5% Triton X-100 and stratified 2 to 5days at 48C before planting. Kanamycin-resistant seedlings were se-lected on half-strength Murashige and Skoog (MS) media (Murashigeand Skoog, 1962) containing 100 mg/mL kanamycin sulfate, plus 2%sucrose where noted. Stock solutions of 2,4-diphenoxybutyric acid(2,4-DB; Sigma) were prepared in dimethyl sulfoxide.

Isolation of the AIM1 Gene

DNA was prepared from aim1 heterozygous plants, using a hexade-cyltrimethylammonium bromide protocol (Murray and Thompson,1980). A genomic library was constructed and screened using stan-dard techniques (Sambrook et al., 1989). Wild-type genomic cloneswere isolated from a Landsberg erecta library (Arabidopsis BiologicalResource Center [ABRC], Columbus, OH; Voytas et al., 1990). A size-selected cDNA library (ABRC; Kieber et al., 1993) was screened forfull-length clones.

DNA Sequencing

Sequencing of larger clones was performed by making serial dele-tions using an ExoIII nuclease–based procedure (Erase-A-Base;Promega, Madison, WI). cDNA clones were sequenced on bothstrands, whereas single-pass one-strand sequencing was used withgenomic clones to identify intron/exon boundaries. The DNA se-quences for AIM1 and AtMFP2 have been deposited in Genbank asaccession numbers AF123253 and AF123254, respectively.

DNA and RNA Gel Blot Analyses

RNA was isolated using the protocol of Chomczynski and Sacchi(1987). Five micrograms of total RNA was loaded on 0.8% agarosegels with 1.1% formaldehyde. RNA gel blot hybridization was per-formed at 688C in modified Church buffer (1 mM EDTA, 0.25 MNa2PO4, 1.0% casein, and 7.0% SDS, pH 7.4). DNA gel blot hybrid-ization was performed using standard techniques (Sambrook et al.,1989).

b-Oxidation Enzyme Assays

Oligonucleotides were designed to construct a full-length AIM1 pro-tein coding sequence suitable for overexpression in a bacterial hostsystem. A BamHI site was engineered 59 of the AIM1 start codon toplace it in frame in pGEX4T-1, a glutathione S-transferase fusionvector (Pharmacia). Protein expression was induced with 0.1 mMIPTG, and the fusion protein was purified by batch method usingglutathione–agarose beads. A NotI fragment from an AtMFP2 cDNAclone, containing the entire coding sequence, was excised and li-gated into pGEX4T-2 and used to produce recombinant protein asdescribed above. The resulting proteins were .95% pure (data notshown). Hydratase activity was measured by following the decreasein absorbance at 263 nm due to the hydration of the double bond ofcrotonyl-CoA (Binstock and Schulz, 1981). Kinetic parameters (Vmax

and Km) were estimated using nonlinear regression on data col-lected (n 5 2) using crotonyl-CoA concentrations ranging from 5 to200 mM.

Fatty Acid Methyl Ester Analysis

Fatty acid composition of total lipids was determined as describedby Browse et al. (1985).

Construction of the Cauliflower Mosaic Virus 35S–Antisense AIM1 Gene Construct

The b-glucuronidase (GUS) cassette from the plant transformationvector pBI121 (Clontech, Palo Alto, CA) was excised using BamHIand SacI and replaced with a full-length AIM1 cDNA clone in the an-tisense orientation. This construct was transformed into Agrobacte-rium tumefaciens GV1301 and introduced into Arabidopsis ecotypeWassilewskija (Ws) by using the vacuum infiltration protocol ofBechtold et al. (1993).

1922 The Plant Cell

Construction of the AIM1–GUS Chimeric Gene and Histochemical Localization of GUS

The putative AIM1 promoter was amplified using a 59 upstreamprimer and a 39 primer with an engineered BamHI site. By using a na-tive XhoI site and the engineered BamHI site, a 2-kb fragment con-taining the first 30 amino acids of the AIM1 protein was ligated intopBI101.2 (Clontech), resulting in an in-frame fusion with the GUS re-porter gene. This construct was transformed into Arabidopsis, as de-scribed above.

Histochemical localization of GUS was performed as described byCraig (1992). For light microscopy, the procedure below was fol-lowed, and tissue sections were visualized using dark-field optics.

Light and Electron Microscopy

All shoot apices were harvested when the primary shoot was .3 cmin length. For light microscopy, samples were fixed overnight in 4%paraformaldehyde or in FAA (4% paraformaldehyde, 50% ethanol,and 5% acetic acid) and taken through a graded ethanol series to100%. Samples embedded in London Resin (LR) White (Electron Mi-croscopy Sciences, Fort Washington, PA) were taken through agraded LR White/ethanol series with several changes at 100% LRWhite. Resin was polymerized according to the manufacturer’s rec-ommendations. Samples embedded in wax (Paraplast Plus; OxfordLabware, St. Louis, MO) were prepared as described by Hensel et al.(1994). Samples for electron microscopy were fixed in FAA and fur-ther prepared as described by Hensel et al. (1994). In situ hybridiza-tion was performed as described by Lincoln et al. (1994).

ACKNOWLEDGMENTS

This work was funded by grants from the National Science Founda-tion (Grant No. DMB-9005164), National Institute of Aging (Grant No.5F32AGO5542-02), and the U.S. Department of Energy/National Sci-ence Foundation/U.S. Department of Agriculture CollaborativeResearch in Plant Biology Program (Grant No. BIR92-20331). T.A.R.has been supported by a National Science Foundation Graduate Fel-lowship, a University of Wisconsin–Madison Genetics DepartmentTraining Grant (National Institutes of Health Grant No. GM07133-181), and a Wisconsin Alumni Research Foundation Graduate Fel-lowship. Special thanks to Sara Patterson, who helped with thesectioning and the microscopy, and to Michelle Nelson, who sup-plied several of the scanning electron microscope (SEM) photographs.We are grateful to Claudia Lipke for photographic work, Heide Barnhillfor help with the SEM work, and Eric Schaller for comments on themanuscript.

Received February 9, 1999; accepted August 9, 1999.

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DOI 10.1105/tpc.11.10.1911 1999;11;1911-1923Plant Cell

Todd A. Richmond and Anthony B. Bleecker-Oxidation Causes Abnormal Inflorescence Development in ArabidopsisβA Defect in

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