A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket

Authors

  • Anders S. Carlsson,

    1. Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA
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    • Present address: Department or Plant Breeding Research, Swedish University of Agricultural Research, S-268 31 Svalöv Sweden.

  • Samuel T. LaBrie,

    1. Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA
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    • Present address: Incyte Pharmaceuticals, 3174 Porter Drive, Palo Alto, CA 94304, USA.

  • Anthony J. Kinney,

    1. DuPont Experimental Station, Wilmington, DE, USA;
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  • Penny Von Wettstein-Knowles,

    Corresponding author
    1. Genetics Department, Molecular Biology Institute, University of Copenhagen, Oester Farimagsgade 2A, DK-1353 Copenhagen K, Denmark.
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  • John Browse

    Corresponding author
    1. Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA
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*For correspondence (fax +45 35322113; e-mail knowles@biobase.dk; fax +1509-335-7643; e-mail: jab@wsu.edu).

Summary

The fab1 mutant of Arabidopsis is partially deficient in activity of β-ketoacyl-[acyl carrier protein] synthase II (KAS II). This defect results in increased levels of 16 : 0 fatty acid and is associated with damage and death of the mutants at low temperature. Transformation of fab1 plants with a cDNA from Brassica napus encoding a KAS II enzyme resulted in complementation of both mutant phenotypes. The dual complementation by expression of the single gene proves that low-temperature damage is a consequence of altered membrane unsaturation. The fab1 mutation is a single nucleotide change in Arabidopsis KAS2 that results in a Leu337Phe substitution. The Leu337 residue is conserved among plant and bacterial KAS proteins, and in the crystal structures of E. coli KAS I and KAS II, this leucine abuts a phenylalanine whose imidazole ring extends into the substrate binding cavity causing the fatty acid chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild-type, the Leu207Phe protein showed a 10-fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size-exclusion chromatography and was severely impaired in condensation activity. These results suggest that the molecular defect in fab1 plants is a structural instability of the KAS2 gene product induced by insufficient space for the imidazole ring of the mutant phenylalanine residue.

Introduction

The biosynthesis of fatty acids is accomplished by a complex of soluble proteins called fatty acid synthase that reiteratively adds C2-units to a fatty acyl chain and prepares the chain for the next round of elongation. Joining eight or nine C2-units together yields the C16 and C18 fatty acids that characterize membrane lipids found in all eukaryotes. In plants and many bacteria a discrete gene encodes each fatty acid synthase component. Isozymes exist for several of the components, among which is the β-ketoacyl [acyl carrier protein] synthase (KAS) enzyme which creates new carbon-carbon bonds when joining the C2-unit to the growing acyl chain. Several well characterized KAS isozymes differ with respect to substrate specificities, sensitivity to the antibiotic cerulenin, whether the primer substrate is activated by acyl carrier protein (ACP) or coenzyme A (CoA) and in the nature of the molecular mechanism of the reaction. In plant plastids and E. coli, KAS III catalyses the condensation of C2-CoA to C4 and is insensitive to cerulenin. KAS I and KAS II use acyl-ACP primer substrates. In plastids KAS I prefers C4- to C14-ACP substrates whereas KAS II has highest activity towards C16-ACP. The former is very sensitive to cerulenin, and the latter moderately so. The reaction catalysed by the KAS enzymes is a Claisen condensation which consists of three steps – transfer, decarboxylation and condensation (Figure 1). KAS I and KAS II catalysis depends on the presence of two histidine residues, while KAS III uses a histidine and arginine (von Wettstein-Knowles et al., 2000). KAS III is thus more similar to chalcone synthase than to the KAS I and KAS II family of enzymes.

Figure 1.

The Claisen condensation carried out by β-ketoacyl [acyl carrier protein] synthase (KAS) I and II consists of three steps.

(a) Transfer of the acyl primer substrate from the pantetheine arm of ACP to the active site cysteine of KAS.

(b) Decarboxylation of the donor malonyl-ACP substrate to give an acetyl-ACP carbanion.

(c) Condensation of the carbanion with the C1 carbon of the acyl primer substrate to form the elongated β-ketoacyl-ACP product.

The pool of 16 : 0-ACP formed by the action of KAS III, KAS I and other enzymes of fatty acid synthase lies at the first branch point in plant lipid metabolism. In mesophyll cells of wild-type Arabidopsis, 69% of this pool is elongated to C18 by the action of KAS II (Browse et al., 1986). The vast majority of these C16 and C18 fatty acids are required for chloroplast biogenesis via the eukaryotic and prokaryotic pathways, resulting in a final ratio for C18/C16 of 2.4 in wild-type Arabidopsis leaves (Kunst et al., 1988). The remaining C16 and C18 acyl chains serve as precursors for other vital lipids such as cutin, suberin and waxes. The essential nature of KAS gene products reduces the chance of identifying viable mutations in the involved enzymes. Nevertheless, the observation that fab1 plants exhibit a decreased C18/C16 ratio (to a value of 1.3) implies a partial blockage in elongation of 16 : 0-ACP. Enzyme assays demonstrated that KAS II activity in partially purified preparations from fab1 plants was only 60% of the activity measured in preparations from wild-type (Wu et al., 1994). These results are consistent with the fab1 lesion being in the gene KAS2 that encodes the KAS II enzyme.

Relative to wild-type, fab1 plants contain increased proportions of 16 : 0 in all the major leaf glycerolipids. In particular, the major chloroplast phospholipid, phosphatidylglycerol (PG) contains 45% 16 : 0 (Wu et al., 1994). As a result the proportion of high-melting-point fatty acids (16 : 0 + 18 : 0 + 16 : 1-trans) is 69% compared with only 55% in wild-type Arabidopsis. Levels of high-melting-point fatty acids above 60% in PG have been correlated with chilling sensitivity in plants. In actuality, fab1 plants do not exhibit classic chilling sensitivity (Wu and Browse, 1995) and only show symptoms of damage after prolonged exposure to low temperature (Wu et al., 1997). Because other evidence points to a role for PG in plant temperature responses (Murata et al., 1992; Wolter et al., 1992), it is tempting to accept that low-temperature symptoms in fab1 plants are consequences of the increased 16 : 0 in PG (and possibly other membrane lipids). However, genetic analyses carried out on fab1 mutants cannot completely exclude the possibility that damage at 2°C results from a second mutation closely linked to the fab1 locus.

To address both of these questions, we undertook further characterization of the fab1 mutant and the KAS2 gene. A putative KAS2 cDNA from Brassica napus complemented the fatty acid defect. The transgenic plants were able to survive low-temperature treatment, thus demonstrating that high 16 : 0 is indeed the underlying cause of the fab1 phenotype. We also identified the Arabidopsis KAS2 ortholog and sequenced the fab1 allele. A single basepair change in the DNA sequence encodes for a Leu337Phe substitution in the KAS II protein. Introduction of the corresponding mutation, Leu207Phe, into the E. coli KAS I protein greatly reduced enzyme activity. This result, in conjunction with the crystal structures of E. coli KAS I and KAS II proteins provided insight into the consequences of the mutation for the three partial reactions.

Results

Expression of a Brassica KAS2 cDNA in the fab1 mutant

A potential soybean KAS2 cDNA was isolated from a developing soybean seed cDNA library using a degenerated oligonucleotide probe designed to a sequence conserved in KAS enzymes. The deduced open reading frame of the resulting cDNA had only 48% sequence identity with that of GmKAS1. Antisense expression of the cDNA in transgenic soybean somatic embryos reduced KAS II activity (Kinney, 1996) and resulted in an increase of 16 : 0 in the total fatty acids from approximately 15% (14.5% ± 1.4 n = 5) in control lines to nearly 40% (38.8% ± 0.8 n = 5). These results indicate that this cDNA, designated GmKAS2, encodes a KAS II enzyme. A BnKAS2 cDNA was identified in a Brassica napus cDNA library using GmKAS2 as a probe. The protein encoded by BnKAS2 has 78% sequence identity to GmKAS2 cDNA but only 49% to GmKAS1. Plasmid pBnKAS2S containing the B. napus KAS2 cDNA under control of the 35S promoter was transformed into fab1 plants. Seventy kanamycin resistant plants were selected and their total leaf fatty acid compositions were determined. The 16 : 0 content of these T1 transformants varied from 6% (wild-type = 12%) to 23% (fab1 = 20%). This difference in 16 : 0 content was presumably due to variability in expression of the BnKAS2 cDNA. Several independent lines with low levels of 16 : 0 showed segregation of the kanamycin marker in the T2 generation (3 resistant : 1 sensitive) indicating the presence of a single T-DNA insert in each. Line 6–24 was characterized in detail.

The overall leaf fatty acid compositions of T2 progeny in line 6–24 fell into two classes. Approximately one quarter of the plants had 16 : 0 and 16 : 3 fatty acid levels comparable to those of the parental fab1 line. The remaining individuals exhibited a 40% reduction in both 16 : 0 and 16 : 3 fatty acids thus containing slightly lower levels of C16 fatty acids than wild-type (Table 1). These results are consistent with complementation of the fab1 defect in plants of line 6–24 that were either homozygous or hemizygous for the pBnKAS2S construct. RNA gel-blot analysis using a radioactive probe specific to the BnKAS2 sequence demonstrated that transcript was present only in segregants containing low C16 fatty acids and not in high C16 segregants or in wild-type controls (data not shown). Each of the major glycerolipids from complemented plants had equivalent or lower proportions of 16 : 0 as those from wild-type. In particular, the level of 16 : 0 in PG was reduced from 51% in fab1 plants to 35%. Thus, while PG from fab1 plants contains approximately 70% high-melting-point fatty acids (Wu and Browse, 1995), PG from the complemented plants contains 57% of these fatty acids compared with 55% for wild-type.

Table 1.  Fatty acid composition of total leaf lipids in T2 plants of line 6–24. The composition for two classes of T2 plants (low C16 and high C16 fatty acid content) are compared with fab1 and wild-type controls grown in the same experiment. Data are means ± sd
Fatty
Acid
6–24 T2
High C16
(n = 7)
6–24 T2
Low C16
(n = 32)
Wild Type
(n = 12)
fab1
(n = 12)
16 : 020.2 ± 2.311.4 ± 1.311.8 ± 0.917.2 ± 2.5
16 : 14.5 ± 0.43.1 ± 0.32.9 ± 0.54.0 ± 0.3
16 : 21.0 ± 0.20.6 ± 0.20.7 ± 0.31.2 ± 0.1
16 : 317.3 ± 1.511.8 ± 2.118.0 ± 2.020.8 ± 3.6
18 : 00.5 ± 0.40.9 ± 0.40.5 ± 0.30.3 ± 0.2
18 : 12.8 ± 0.93.8 ± 0.82.3 ± 0.82.1 ± 0.5
18 : 29.0 ± 0.614.5 ± 2.411.8 ± 2.19.4 ± 1.2
18 : 344.6 ± 2.353.9 ± 1.952.0 ± 1.745.1 ± 2.0
 
Total C1643.026.933.443.2

To investigate the relationship between high 16 : 0 and the low-temperature phenotype, we randomly chose 36 T2 progeny of line 6–24. DNA was prepared from a single leaf of each plant and a PCR reaction was used to test for the presence or absence of the pBnKAS2S construct. A second leaf was used to determine fatty acid composition before transferring the plants to 2°C. After 4 weeks at 2°C, it was easy to distinguish those plants that had survived the low-temperature treatment from those that exhibited the fab1 phenotype. Twenty-eight of the 36 plants were positive for the pBnKAS2 transgene by PCR (Figure 2). All of these plants were as healthy as wild-type controls at the end of the 4-week cold treatment and all contained less than 31% C16 fatty acids in their total leaf lipids. By contrast, eight plants lacking the pBnKAS2 construct were severely damaged or killed by treatment at 2°C and contained more than 38% C16 in their total leaf lipids. Thus, phenotypic observations infer that complementation of the high C16 character of fab1 plants also results in complementation of their low-temperature susceptibility. A quantitative measure of the damage to fab1 plants at 2°C can be made by assaying photosynthetic capacity by fluorescence analysis (Wu et al., 1997). After 4 weeks at 2°C, the eight T2 segregants lacking the pBnKAS2 construct and exhibiting high C16 fatty acids provided an average ratio of Fv/Fm of 0.10 as did fab1 controls, consistent with previous studies of the fab1 mutant (Wu et al., 1997). By contrast, segregants containing pBnKAS2 and having low 16 : 0 had an Fv/Fm of 0.70 compared with 0.69 for wild-type controls in the same experiment. The successful transgenic complementation of the low- temperature phenotype precludes the possibility that damage in fab1 plants results from a second mutation that is tightly linked to the fab1 locus.

Figure 2.

Segregation of the BnKAS2 transgene, chilling phenotype, and C16 fatty acid content in a T2 population of the transgenic line 6–24.

PCR reactions using DNA from 36 T2 plants were analysed on an agarose gel. T2 plants carrying the transgene are indicated by the presence of an approximately 1.3 kb PCR product.

The same plants were scored for survival at 2°C (+, tolerant; –, sensitive) and C16 to C18 fatty acid ratio (H = high, L = low).

Characterizing the Arabidopsis KAS2 gene

The BnKAS2 cDNA was used to screen 4 × 104 plaque forming units of a λgt11 library derived from Arabidopsis genomic DNA (ecotype WS), and nine candidate clones were selected. PCR identified clone TK94b of which 6972 nucleotides were sequenced as described in Experimental procedures plus the text of Figure 3. Comparing the genomic sequence with that of available KAS2 cDNAs revealed the presence of 11 well-defined introns and suggested that another must occur in the transit peptide. To determine which of the nine possible splice sites encompassed this putative intron and to define the amino terminal end of the transit peptide, RT–PCR was carried out on total RNA. Sequencing led to identification of the missing intron and revealed two possible ATG start codons. By comparison to start codons of other KAS2 genes (Slabaugh et al., 1998) and the 5/9 identical residues to the consensus initiation region for plant genes (Fütterer and Hohn, 1996) versus 3/9 for the second ATG, the upstream one is considered to be the more likely translation start site

Figure 3.

The structure and sequence of the Arabidopsis gene encoding β-ketoacyl-ACP synthase II.

(a) Exon/intron map of the KAS2 genomic region. Closed bars represent exons and open bars introns whose lengths in bp are specified. ATG and TGA indicate the sites of the first methionine codon and the stop codon, respectively. The bar denoted F600 covers the region amplified by the core primers that was used to identify the KAS2 genomic clone.

(b) Nucleotide sequence of KAS2 (GenBank accession no. AF318307) and its deduced amino acid sequence. The mutated base in fab1 is in bold and its codon underlined. The asterisk indicates the active site cysteine, and the vertical arrow the predicted transit peptide cleavage site. Primers used to amplify fragments for sequencing are shown by arrows and numbered: 1 and 2 represent the core set, 3 and 4 the N-terminal set and 5 (arrow in intron 3, see a) and 6 the C-terminal set. Primer sequences are given in Materials and methods.

The structure of the Arabidopsis KAS2 gene is schematized in Figure 3a. The KAS2 cDNA contains 1626 bp encoding a 541 residue protein derived from 13 exons and 12 introns that extend over 3322 bp. By comparison to the known amino terminal sequence of barley KAS I (Siggaard-Andersen et al., 1991) and the expected size of mature KAS II proteins from immunoblots of extracts using KAS II antibodies (Slabaugh et al., 1998), a transit peptide cleavage site occurs after amino acid 122 resulting in a mature KAS II protein of 419 residues. KAS II has 56% sequence identity to Arabidopsis KAS I (Millar and Kunst, 1995) and a predicted identity of 40% (von Wettstein-Knowles et al., 2000) to a putative mitochondrial KAS (mt-KAS) (Mekhedov et al., 2000). The origin of these three proteins, however, appears quite diverse as KAS I has 7 introns and mt-KAS 13. Only introns 4 in KAS I and 12 in mt-KAS are localized identically The predicted transit peptide sequences are also quite divergent (see also Slabaugh et al., 1998).

The WS wild-type sequence in TK94b extends 1803 bp upstream from the ATG start codon and 1782 downstream from the TGA stop codon. A comparison to the analogous Columbia sequence in chromosome 1 BAC F9E10 reveals a difference of only 2 bp. At −531 WS has a C versus a T in Columbia, while at 1336 in the middle of the third intron WS has a G and Columbia an A.

The KAS2 allele in fab1 differs by a single bp that influences catalytic activity

Genomic DNA from the fab1 line encompassing KAS2 (−249 bp upstream to +431 bp downstream) was sequenced. A single bp difference was identified, namely thymine in place of cytosine at bp 1009, giving rise to the mutation Leu337Phe, that is the likely basis of the genetic lesion in fab1 plants.

Expression of plant KAS I and KAS II proteins in E. coli yields insoluble protein unsuitable for biochemical characterization (Chuck et al., 1995; Wissenbach et al., 1995). Therefore, to ascertain the importance of the leucine residue, an analogous mutation was introduced into the E. coli fabB and fabF genes encoding KAS I and KAS II, respectively, whose acyl binding pockets are so similar that substrate specificities can not be attributed solely thereto. Both the cloned wild-type and mutant genes were expressed in E. coli. Expression of the Leu208Phe mutant of fabF led to insufficient soluble protein for analysis revealing a detrimental effect of this residue on KAS II stability. Thus KAS I and Leu207Phe proteins were assayed for their ability to carry out the three partial reactions of the Claisen condensation (Figure 1).

In the initial step a fatty acid is transferred from ACP to KAS resulting in formation of a fatty acid-KAS complex (Figure 1). The reaction can be followed and quantitated using radiolabelled myristic acid and resolving the acylated-ACP substrate and acylated-KAS product on a Superdex-200 size exclusion column. When this was done with the Leu207Phe protein, the acyl-KAS eluted as a much broader peak shifted toward a lower molecular weight when compared to wild-type. The acyl transfer reaction can be considered as formation of an enzyme substrate complex, namely [acyl-ACP-KAS], for which the initial rate can be measured as a function of one of the substrates (k1[S]) giving half saturation values that equal Kd (McGuire et al., 2001). For the wild-type KAS I 1/k = 1.9 µm−1 which gives a binding affinity of 5 × 10−7 M for KAS I (0.1–9.0 µm) to C14 (0.225 µm). The Leu207Phe KAS, 1/k = 0.12 µm−1 had a 10-fold lower binding affinity, namely 8.3 × 10−6 m for KAS I (0.5–15.2 µm) to C14 (0.225 µm). The second step, in which the KAS protein decarboxylates the donor substrate malonyl-ACP to the acetyl-ACP carbanion (Figure 1), was monitored using a radiolabelled substrate and analysing the TCA precipitated substrate and product on 1 m urea-13.3% polyacrylamide gels. At higher protein concentrations (4 µm) Leu207Phe was almost as efficient as the wild-type, while at lower concentrations (0.4 µm) neither loss of substrate nor formation of acetyl-ACP carbanion was detected after 30 min. The wild-type was active at 0.2 µm in 20 min assays. The final or condensing step (Figure 1) is assayed by determining whether a KAS can restore elongation activity to a crude E. coli protein extract treated with cerulenin (CIPE) that binds covalently to the active site cysteine (Funabashi et al., 1989; Kauppinen et al., 1988). Radiolabelled malonyl-CoA and a KAS protein are added to a CIPE preparation and the resulting acyl-ACPs separated using 4 m urea-13.3% polyacrylamide gels, as exemplified in Figure 4. The first two lanes reveal the effect of cerulenin on the ability of the crude protein extract to elongate fatty acids. Addition of wild-type KAS (lane 3) complements the inhibition caused by cerulenin as exhibited by the presence of the same acyl-ACPs observed in the non-cerulenin-treated protein extract (lane 1). Addition of Leu207Phe KAS does not restore synthesis of the longer acyl-ACPs (lane 4) even though twice as much mutant as wild-type KAS was added.

Figure 4.

Assay of E. coli of wild-type and Leu207Phe KAS I proteins in cerulenin inhibited protein extracts (CIPE).

The 100 µl reaction was carried out by incubating 60.1 µm[2–14C]-malonyl-CoA (1670 dpm/nmol), 10 µm ACP, 10 µm acetyl-CoA, 59 mm K-PO4 pH 6.8 buffer, and CIPE with either KAS I or Leu207Phe protein (0.25 or 0.14 µm, respectively) for 20 min at 37°C (McGuire et al., 2001).

The acyl-ACPs were precipitated, resuspended and after resolution by conformationally sensitive 13.3% acrylamide, 4 m urea gels, results were visualized by blotting to a PVDF membrane and autoradiography. –, untreated control; +, CIPE control; WT, CIPE with wild-type KAS I; L207F, CIPE with mutant KAS I.

The acyl chains of some of the acyl-ACP species inferred by comparison to standards are shown.

Discussion

A Brassica KAS2 cDNA complements both fatty acid composition and chilling sensitive phenotype of fab1

Previous studies of the fab1 mutant established two phenotypes. The first is a high total content of C16 fatty acids in leaves, roots and seeds (Wu et al., 1994). The second is a sensitivity to low temperatures, which leads to damage and death of fab1 plants (Wu et al., 1997). While co-segregation analysis indicated a relationship between the increase in C16 fatty acids and the low-temperature phenotype, the possibility could not be eliminated that a closely linked gene had been mutated simultaneously in the mutagenesis protocol yielding fab1. In the present study we have eliminated this possibility by transforming a Brassica KAS2 cDNA into the fab1 mutant and demonstrating that both phenotypes are complemented in the resulting transgenic plants. Thus the low-temperature damage is a pleiotropic effect resulting from the fab1 mutation and the resultant increase of 16 : 0 in membrane lipids.

Nature of the molecular defect in KAS II from fab1 plants

The original suggestion that the change in fatty acid composition characterizing fab1 was attributable to a mutation of the KAS2 gene was based on the observation that KAS II activity in extracts of fab1 plants was 60% of that in wild-type extracts. To confirm this suggestion, the KAS2 allele in the fab1 mutant strain was sequenced, identifying a single bp change resulting in Leu337Phe. Comparison to the plant KAS I and KAS II sequences in GenBank reveals that this leucine is part of the conserved tripeptide ALS (Figure 5). The ALS sequence also occurs in the putative mt-KAS from Arabidopsis (Mekhedov et al., 2000), KAS I and KAS II from E. coli as well as from a number of other bacteria. Based on sequence comparisons the plant enzymes appear to have been derived from an E. coli-like KAS II progenitor (Olsen et al., 1999). Combining these observations suggested that introduction of the Leu337Phe mutation in the E. coli genes, followed by their expression, offered an avenue to study the mutation's effect on catalysis and stability. Such analyses with the E. coli KAS I and its mutant Leu207Phe revealed the following: (1) The Leu207Phe KAS was inactive in the condensation assay at the tested protein concentration, which was twice that of the wild-type. (2) At high protein concentrations the decarboxylation step was carried out by Leu207Phe almost as well as by the wild-type suggesting that the block in condensation did not result from a serious defect in this activity. (3) Transfer of the acyl substrate to Leu207Phe KAS was markedly inferior to transfer to wild-type KAS I implying that the inability to bind substrate in the active site was a major contributor to the condensation block.

Figure 5.

The Arabidopsis KAS II amino acid sequence compared to those of E. coli KAS I and KAS II (GenBank # M24427 and # Z34979, respectively).

The mutated residue in fab1, Leu337 is part of the conserved sequence ALS (asterisks), and corresponds to Leu207 in E. coli KAS I and Leu208 in E. coli KAS II. Black highlights identical residues, gray conservative substitutions. Dots indicate gaps introduced to maximize alignment.

Crystal structures of the E. coli KAS I and II enzymes (Huang et al., 1998; Olsen et al., 1999) reveal that they are homodimeric enzymes, each subunit of which is composed of a core domain formed by the thiolase α-β-α-β-α fold from which a capping domain protrudes. The structures of the inhibitor cerulenin complexed with KAS II and of C10 and C12 fatty acyl substrates with KAS I are also available (Moche et al., 1999; Olsen et al., 2001). In the KAS I structure, the imidazole ring of Phe201 extends into the substrate binding pocket and causes the fatty acid to bend in the shape of a U at carbons 6–8. Leu207 is in contact with Phe201 and modelling suggests that the imidazole ring of Phe201 in the Leu207Phe mutant will not have adequate space unless structural adjustments are made by the enzyme. In the E. coli KAS II enzyme, the same structural considerations apply to residues Phe202 and Leu208. We suggest that a comparable steric clash is the basis of the fab1 mutant phenotype.

Activity of the EcKAS I enzyme is severely impaired by the Leu207Phe mutation. This result, plus the conservation of the equivalent leucine residue within the KAS I-KAS II family, suggest that the Leu337Phe mutation in AtKAS II should markedly compromise the enzyme activity of the protein. Nevertheless, the 16 : 0-ACP elongation activity measured in extracts from fab1 plants was 60% of the wild-type activity, consistent with the C18 fatty acid content of fab1 plants (Wu et al., 1994). The probable explanation for these observations is that the effect of the Leu337Phe change on AtKAS II activity is much less drastic than that of the analogous Leu207Phe mutation in EcKAS I because of other subtle structural differences in the enzymes. This suggestion is supported by the fact that arsenite very effectively inhibits C16 to C18 elongation (Harwood and Stumpf, 1972; Mikkelsen and von Wettstein-Knowles, 1978) which precludes the possibility of another isozyme carrying out the same reaction. The recently completed Arabidopsis genome sequence, moreover, does not appear to encode a second KAS II isozyme.

Experimental procedures

Plant materials and growth conditions

The fab1 mutant in line 1A9 of Arabidopsis thaliana (L) Heynh descended from the Columbia wild-type (James and Dooner, 1990) was backcrossed to wild-type four times. Plants were grown on commercial potting mix at 22°C under continuous fluorescent illumination (150 µmol m-2 sec-1) and 60–70% relative humidity. 15-day-old-plants were transferred to 2°C under continuous light for up to 4 weeks, during which time they were monitored for changes in growth and development. Mutants used for in planta transformation were grown in continuous light at 22°C for 4 weeks. To stimulate growth of secondary bolts, primary bolts were trimmed away approximately one week prior to infiltration.

Embryos, 20–21 days after pollination, from Brassica napus (variety Westar) and those of 50 mg from soybeans Glycine max (variety Wye) were used for mRNA isolation (Yadav et al., 1993).

Cloning a Brassica napus KAS2 cDNA for screening and transformation

Potential soybean GmKAS2 cDNA clones were isolated by screening a λ-Zap (Stratagene) cDNA library (Yadav et al., 1993) from developing soybean seed using a degenerate oligonucleotide probe designed to the peptide sequence, CDAYHMTDP, following the λ-ZAP Cloning Kit Manual (Stratagene). The probe was based on a conserved region of KAS I clones from soybean, Brassica and barley (GenBank # AF243182, AF243183, M60410) and E. coli KAS I (# M24427, Z34979). The primary clones selected were then screened directly on λ DNA by PCR using primers based on the conserved KAS regions: CDAYHMTDP and RRVVTGMG. One of the identified clones, designated GmKAS2, was used to screen a cDNA library made from developing Brassica seeds in λ-Zap (Yadav et al., 1993). This identified a potential Brassica BnKAS2 cDNA. 2117 bp of the latter was inserted into the EcoRI site of the binary Ti plasmid, pZS199 (Browse et al., 1993), giving rise to plasmid pBnKAS2S with the BnKAS2 cDNA under transcriptional control of the cauliflower mosaic virus 35S promoter. As selectable markers for transformation pZS199 contains the chimeric gene nopaline synthase/neomycin phosphotransferase (Bevan, 1984) for plant cells, and the bacterial neomycin phosphotransferase gene from Tn5 (Berg et al., 1975) for Agrobacterium tumefaciens. The nopaline synthase promoter was replaced by the 35S promoter (Odell et al., 1985). The binary vector pBnKAS2S was introduced into A. tumefaciens GV3101 by the freeze thaw method (Holsters et al., 1978). Transformed clones were identified on Luria Bertani-agar plates containing 50 µg ml−1 kanamycin and gentamycin.

Production of fab1 transformants

Using vacuum infiltration fab1 mutants were transformed with A. tumefaciens GV3101 harboring the plasmid pBnKAS2S, as described by Tokuhisa et al. (1998). T1 seeds were surface sterilised (Clough and Bent, 1998), and screened on plates containing 1/3 × MS medium (Sigma Chemicals), 0.8% agar (Sigma) and kanamycin monosulfate 50 µg ml−1. Resistant T1 plants were transferred to soil. Seeds from individuals having a reduced content of C16 fatty acids were planted, and the T2 progeny tested for kanamycin resistance and leaf fatty acid composition.

DNA and RNA manipulations

The λgt11 clone TK94b was amplified (Sambrook et al., 1989) from a Wassilewskija (WS) genomic DNA library (Yadav et al., 1993). PCR was carried out using AmpliTaq (Perkin Elmer) and three overlapping sets of primers to amplify most of the wild-type mature KAS II sequence in TK94b. (1) The core primers specific for KAS2 genes (Wissenbach, 1994): sense, 5′-TGGGTIGCICCIAAR YTIWSIAA-3′; antisense, 5′-GCRCAIGCIGTIGAIATIGARTARTT-3′. (2) N-terminal primers: redundant to conserved region VVVTGMG sense, 5′-GTIGTIRTCACIGGIATGGG-3′; KAS2 antisense, 3′-CAA GCAGTTGAAATAGAATAGTT-5′. (3) C-terminal primers: KAS2 intron 3 sense, 5′- CTGACAAAAGAGTCACATG-3′, redundant to conserved region NSFGFGG antisense, 5′-CCICCRAAICCRAAI SWRTT-3′. The obtained fragments were cloned, sequenced and the sequence extended by walking using appropriate primers and TK94b as template. Genomic DNA isolated from fab1 mutants (Aldrich and Cullis, 1993) was used to amplify fragments of the KAS2 gene with the aid of appropriate primers. The fragments were directly sequenced. Both strands were completely sequenced in all cases.

To test for the presence of the transgene, genomic DNA was isolated from wild-type, fab1, T1 and T2 plants. The DNAs were used as templates for PCR with the primers: sense, 5′-ATT CTCTCTTCACCCTTTTCTC-3′; antisense, 5′-AATACACAGAAT CACACCAG-3′ that amplify the KAS2 gene from BnKAS2 cDNA from −91 to 1221.

Probes were 32P random labelled (Prime-a-Gene Labelling System, Promega). They included the BnKAS2 cDNA for initial screening of the λgt11 library, F600 for identifying the KAS2 containing clone, and a 260-bp fragment from the 3′non-coding region of BnKAS2 cDNA for identification of the transgene BnKAS2 in total mRNA.

Total RNA was isolated from Columbia and T2 plants from the transgenic line 6–24 using TRIzol (Gibco BRL). Columbia RNA was subjected to reverse transcriptase PCR (SuperScript One-StepTM. RT–PCR System, Gibco RBL), and the resulting fragments sequenced in both directions. To verify the start site of the KAS2 mRNA PCR was carried out on total RNA using primers to the most amino terminal sequence. To confirm expression of the transgene, 20 µg RNA from the T2 plants and Columbia was subjected to the NorthernMax protocol (Ambion) and hybridized to the random labelled 260 bp 3′ non-coding sequence of the BnKAS II cDNA using ULTRAhyb hybridization buffer (Ambion) at 42°C. The blot was washed 2 × 25 min at 42°C in high stringency wash solution #2 (Ambion).

Fatty acid, lipid and fluorescence analyses

The overall fatty acid composition of leaves and individual lipids was determined as described (Wu et al., 1994). Samples (1 µl) of the organic phase were analysed by GC on a 30-m X 0.53-mm Alltech Econo-cap column containing a 1.2-µm EC-WAX phase (Alltech Associates, Inc., Deerfield, IL, USA). The GC was programmed for an initial temperature of 160°C for 1 min, followed by an increase of 20°C min−1 to 190°C and a second increase of 4.5°C min−1 to the final temperature 203°C. Results are expressed as mol percentage.

Measurements of modulated chlorophyll fluorescence after chilling treatment were made (Wu et al., 1997). They enabled calculation of the ratio of instantaneous fluorescence (Fv): steady-state fluorescence (Fm) at 22°C in the dark.

Catalytic activities

To determine the potential effect of the fab1 mutation on catalysis, a model system employing E. coli KAS enzymes was used. The analogue of the Arabidopsis KAS II, Leu337Phe mutation was introduced into the wild-type E. coli genes fabB and fabF via site directed mutagenesis (Siggaard-Andersen et al., 1998; McGuire et al., 2001) using the following primers plus their complements: KAS I, L207P 5′-CGCAATGGGTGCGTTCTCTAC TAAATACAACG-3′; and KAS II, L208P 5′-GCGGCACGTGCA TTCTCTACCCGCAATGATAACC-3′. Wild-type and both mutants were expressed in E. coli leading to synthesis of KAS proteins. These were purified with the aid of an amino terminal His tag, and their ability to carry out the three partial reactions of the Claisen condensation (Figure 1) assayed (McGuire et al., 2001).

Acknowledgements

We are grateful to Katie Dehesh for the pQE30 clone encoding the E. coli fabF gene, and to Johan Gotthard Olsen for modelling phenylalanine in place of leucine 207 in KAS I of E. coli. This work was supported by grants from the Swedish Foundation for International Cooperation in Research and Higher Education (to ACS), the Novo Nordisk Foundation, the Danish Natural Sciences Research Council and the Danish Agriculture and Veterinary Research Council (to PvWK) and the United States National Science Foundation (Grant No. IBN-0084329 to JB).

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