Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean

ARTICLE IN PRESS Journal of Plant Physiology 164 (2007) 1516—1526

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Molecular cloning and characterization of a novel microsomal oleate desaturase gene from soybean Lingyong Li, Xiaolin Wang, Junyi Gai, Deyue Yu Nanjing Agricultural University, National Center for Soybean Improvement, National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing 210095, PR China Received 8 July 2006; accepted 25 August 2006

KEYWORDS FAD2; Microsomal oleate desaturase; Polyunsaturated fatty acids (PUFAs); Soybean (Glycine max Merr. L)

Summary In plants, the endoplasmic reticulum (ER)-associated oleate desaturase (FAD2) is the key enzyme responsible for the production of linoleic acid in non-photosynthetic tissues. In soybean three FAD2-like genes have been reported including two seedspecific genes, FAD2-1A and FAD2-1B, and a house-keeping gene FAD2-2. In this study, we isolated a novel gene encoding FAD2 isoform, designated as FAD2-3. The deduced amino acid sequences of the FAD2-3 displayed the typical three histidine boxes characteristic of all membrane-bound desaturases, and possessed a C-terminal signal for ER retention. Phylogenetic analysis showed that FAD2-3 is grouped within plant house-keeping FAD2 sequences. Yeast cells transformed with a plasmid construct containing the FAD2-3 coding region accumulated a considerable amount of linoleic acid (18:2), normally not present in wild-type yeast cells, suggesting that the isolated gene encodes a functional FAD2 enzyme. Semi-quantitative RT-PCR and in silico analysis showed that FAD2-3 gene is constitutively expressed in both vegetative tissues and developing seeds. In soybean leaves, the level of linolenic acid (18:3) increases with the decrease of linoleic aicd (18:2) under cold treatment. However, no significant change of transcript levels of FAD2-2 and FAD2-3 genes was detected. These results indicated that the altered polyunsaturated fatty acid levels in leaves treated with cold stress have no direct correlation with the expression of these two microsomal oleate desaturase genes. & 2006 Elsevier GmbH. All rights reserved.

Introduction Abbreviations: DAF, days after flowering; ER, endoplasmic reticulum; FAD, fatty acid desaturase; PUFA(s), polyunsaturated fatty acid(s); RACE, rapid amplification of cDNA ends; X:Y, a fatty acyl group containing X carbon atoms and Y cis double bonds Corresponding author. Tel./Fax: +86 25 84396410. E-mail address: [email protected] (D. Yu).

As in all other organisms, fatty acids in plants are the major structure components of membrane phospholipids and triacylglycerol storage oils. The relative quantities of the various saturated and

0176-1617/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2006.08.007

ARTICLE IN PRESS A novel oleate desaturase gene in soybean unsaturated fatty acids (PUFAs) are the major factors influencing the quality of plant oils. For example, oils high in oleic acid (18:1) and low in PUFAs appear to have improved nutritional benefits to human and animal consumption and increased stability (Liu and White, 1992). Fatty acid desaturases (FADs) are enzymes responsible for the insertion of double bonds into fatty acyl chains, following the removal of two hydrogen atoms. These desaturation processes take place in both the plastidial membrane and the endoplasmic reticulum (ER) membrane via two different pathways (Ohlrogge and Browse, 1995). The genes for ER- and plastid-derived D-12 FADs have been characterized from some plant species. Several different microsomal oleate desaturase (FAD2) genes may exist, depending on the particular plant. For instance, there is only one FAD2 gene existing in Arabidopsis (Okuley et al., 1994), and two different FAD2 genes in olive have been identified encoding seedspecifically and constitutively expressed microsomal oleate desaturases (Hernandez et al., 2005). Whereas, three different FAD2 genes have been identified from both cotton and sunflower, with one expressed specifically in seed and the other two expressed in all tissues tested (Liu et al., 1999; Martinez-Rivas et al., 2001; Pirtle et al., 2001). In soybean, one of the most important resources of vegetable oil, two different microsomal oleate desaturase genes have previously been reported: a constitutively expressed gene FAD2-2, and a seedspecific gene FAD2-1; the latter one plays a predominant role in determining the PUFA content of the seed-storage oil (Heppard et al., 1996). A seed-specific isoform of FAD2, designated as FAD21B, has been reported recently in soybean (Tang et al., 2005). However, it is not clear if there exist any other FAD2-like genes in soybean. Thus, in order to explore the regulatory mechanism of oleate desaturation, we isolated a novel microsomal oleate desaturase (FAD2) gene in soybean and demonstrated its function by expression in yeast (Saccharomyces cerevisiae). Meanwhile, the expression pattern of this gene was investigated in different tissues and during soybean seed development.

Materials and methods Plant material and growth conditions Soybeans (Glycine max L. cv Meng8206) were grown in growth chamber with day/night cycle of 32/28, 28/22, 18/12 and 12/8 1C. The light/dark cycle was 12/12 h. Developing seeds were harvested at 11, 14, 19, 20, 22, 24, 27 and 32 days after

1517 flowering (DAF) from field, chilled in liquid nitrogen and stored at 70 1C. Seeds at 22 DAF were dissected out to collect seed coat, embryo and cotyledon, frozen in liquid nitrogen and stored at 70 1C. Leaf, stem, and root tissues were collected from soybean seedlings grown at 28/22 1C with light/dark cycle 12/12 h. Soybean seedlings were grown at 28 1C for 12 days and then shifted to 8 1C, and leaves were collected at 0, 1, 2, 4, 8, 16, 24, 48 h and 1 week.

RNA extraction and RT reactions Total RNA was isolated from different soybean tissues using Plant RNA extraction Kit (TianGen, Beijing) as described by manufacturer. RNA concentration was determined spectro-photometrically and verified by ethidium bromide staining of agarose gel. Total RNA was then treated with RNase-free DNase I (TaKaRa), and about 2 mg was used as template for the first cDNA synthesis using Superscript First Strand Synthesis system and oligo(dT) primers (TaKaRa) according to manufacturer’s protocol.

Isolation of microsomal oleate desaturase partial cDNA clone Two degenerate primers, P1 (50 -AAG AA [AG]G CGA T[ACT] CCG CCG CA[CT]TG-30 ) and P2 (50 -GC [CT]T CCA TGG C[AG] T[GT] [AG]T A[AG] TG-30 ), were designed from the comparison of known plant FAD2 amino acid sequences, corresponding to highly conserved regions (Fig. 1). This pair of primers, together with an aliquot of cDNA from 19 DAF seeds, young leaves, stems and roots, were used in a standard PCR amplification protocol with Hotstart Ex DNA polymerase (TaKaRa). One fragment was generated in each reaction, then subcloned into the vector pGEM-T (Promega) and sequenced.

Rapid amplification of cDNA ends (RACE) The full-length cDNA clone was obtained by 50 and 30 RACE using the SMART RACE cDNA Amplification Kit (BD Biosciences Clontech, Palo Alto, CA) as described by the manufacturer. The gene-specific primers used for RACE were designed from the above partial cDNA sequence. The primers 5R1 (50 AGC CCA ATA GAT TGC CAT GCC ACG-30 ) and 5R2 (50 GGG TGG CAA CAT AAT AGA GGC AGA-30 ) were used for 50 RACE, and the primers 3F1 (50 -CGT ATG ATA GGT TTG CTT CCC ACC T-30 ) and 3F2 (50 -AGA TGC AGG AGT ACT TGC AGT ATG-30 ) were used for 30 RACE. The PCR fragments were cloned into the pGEM-T vector (Promega) and sequenced by Invitrogen (Shanghai).

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Figure 1. Amino acid sequence of FAD2-3 comparison with the microsomal (FAD2-1A, L43920; FAD2-1B, AB188251 and FAD2-2, L43921) and plastidal (FAD6, L29215) homologs from soybean. For alignment, the ClustalX program was used. The conserved amino acids are on gray background. The three His boxes are indicated by subscripts, and the potential membrane-spanning domains are boxed. Solid arrows indicate the regions used for deducing degenerated oligonucleotides for the first PCR amplification. The cDNA sequence of FAD2-3 was deposited in the GenBank database with accession numbers DQ532371.

Expression analysis by semi-quantitative RT-PCR Semi-quantitative RT-PCR analysis was performed using One-Step RT-PCR kit (TaKaRa) with Hotstart

Ex DNA Polymerase (TaKaRa) following the manufacturer’s protocol, and the relative amounts of all RNAs were calculated using GeneQuant Pro RNA/ DNA Calculator (Amersham pharmacia Biotech). Primers for each of the selected genes were

ARTICLE IN PRESS A novel oleate desaturase gene in soybean designed at the length of 20–22 bases, optimized for Tm and GC content using Primer Premier 5.0 software (Palo Alto, CA). The primer sequences used were: FAD2-2, RT2F (50 -TGA AGC GGG TGC CAT TTG-30 ) and RT2R (50 -TGG CAA GAC GGA AAA GGC-30 ); FAD2-3, RT3F (50 -TGT GTG TTT ATG GAG TTC C-30 ) and RT3R (50 -AAC CAT CCC ACC TGA CGG-30 ). As reference, a part of the coding region of the soybean b-Actin gene was amplified with the specific forward primer Actin-1 (50 -TCA CCA CAT CGC CTC AAG-30 ) and the reverse primer Actin-2 (50 -ATC TTC CAC TGC TAC CTG-30 ). PCR was performed using equal amount of template and gene specific or b-Actin primers and carried out for different numbers of cycles in order to optimize reproducibility and ensure that reactions remained in log-linear range. To confirm the specificity of PCR primers for FAD2-2 and FAD2-3, we carried out a negative control 1 using pGEM-T vector containing FAD2-3 cDNA as template with FAD2-2 specific primers. Control 2 was carried out using pGEM-T vector containing FAD2-2 cDNA as template with FAD2-3 specific primers. No corresponding PCR product was observed in these negative controls (data not shown).

ESTs search, assignment to organs and statistical analysis Soybean ESTs were retrieved using the BLASTN program available at TIGR (http://www.tigr.org/ tgi) with predicted open reading frame (ORF) of FAD2-3 as query. To assign these ESTs to the organs from which they originated, the 84 different cDNA libraries were classified and grouped into eight synthetic libraries based on the organ used for construction of each cDNA library. Total numbers of ESTs in the synthetic libraries are: seed, 73,123; root, 44,458; leaf, 35,440; flower, 25,673; seedling, 45,309; stem, 5,795; cell suspension culture, 14,795; mixed tissues, 59,342. For the FAD2-3 gene, the numbers of ESTs in the synthetic libraries (organs) were used to compute the probability of differential expression between all pairs of organs, except for cell suspension culture and mixed tissues, by using online version of the statistical program of Audic and Claverie (1997), http:// igsserver.cnrs-mrs.fr/audic/significance.html.

Expression of FAD2-2 and FAD2-3 in S. cerevisiae The corresponding ORFs of the two soybean FAD2 genes (FAD2-2 and FAD2-3) were amplified by PCR using Hotstart Ex DNA polymerase (TaKaRa) and the following pairs of specific primers: Fad22up

1519 (50 -AAGCTT ACT TTC AGA TTG TGT GTT-30 ) and Fad22down 50 -CTCGAG ATA CTA AAC AGT CAC AAG-30 ) for FAD2-2; and Fad23up (50 -AAGCTT CCT CCT CTT CAC ACA TTT TC-30 ) and Fad23down (50 -CTCGAG GAA AGT TCA AAG AAG CCT CG-30 ) for FAD2-3. For ligation behind the constitutive GAL1 gene promoter of the yeast expression vector pYES2, the primers for FAD2-2 and FAD2-3 were both extended by HindIII and XhoI restriction site (underlined). The resulting PCR products were subcloned into the clone vector pGEM-T, digested with the corresponding restriction enzymes and ligated into HindIII- and XhoI-digested pYES2. The sense orientation of the corresponding inserts relative to the GAL1 promoter was confirmed by restriction mapping and sequencing. These resulting plasmids were called pYES2-FAD2-2 and pYES-FAD2-3, respectively. The S. cerevisiae strain INVSc1 (MATa his3D1 leu2 trp1289 ura3-52/MATa his3D1 leu2 trp1-289 ura3-52, Invitrogen) was transformed with these plasmids by the lithium acetate method and selected on synthetic complete medium lacking uracil. A single colony was grown in synthetic complete minus uracil (SCU) medium containing 2% galactose and 2% raffinose at 30 1C with shaking until stationary phase.

Fatty acid analysis Yeast cultures were harvested by centrifugation (4000 g, 10 min), washed three times with water, and then converted to spheroplasts by enzymatic digestion of cells walls. The fatty acids were extracted and transmethylated with 5% HCL in methanol at 85 1C for 3 h (Pirtle et al., 2001). One-step method described by Garces and Mancha (1993) was used to determine the fatty acid composition of soybean leaves. Following the addition of 13.2 mL of methanol–toluene–dimethoxypropane–H2SO4 (39:20:5:2, v/v/v/v) and 6.8 mL heptane to 300 mg soybean leaves, the mixture was incubated for 1 h at 80 1C, forming a single phase. After cooling, the upper phase containing the fatty acid methyl esters was separated, washed with 5 mL 6.7% Na2SO4, and evaporated to dryness in clean bench. These methyl esters were dissolved in the appropriate volume of heptane and analyzed using GC (model 6890N, Agilent, Palo Slto, CA, USA) with ChemStation software (Agilent, Palo Alto, CA, USA). The GC was fitted with a capillary column (30 m  0.25 mM, 0.2 mm film thickness) of fused silica (Supelco, Bellafonte, PA, USA) and a FID detector. The operating conditions for GC/FID analysis were set as follows: injector 220 1C; FID, 220 1C; flow rate of N2 as a carrier-linear velocity,

ARTICLE IN PRESS 1520 31 cm s1; split ratio, 50:1; flow rate of air, 385 mL min1; H2, 32 mL min1.

Sequence analyses Nucleotide sequence from cDNA clone and the deduced amino acid sequence were identified by the NCBI BLAST program (http://www.ncbi. nlm.nih.gov/BLAST/). Prediction of ORF and theoretical molecular weight of deduced polypeptide were made using the EditSeq program, version 3.88. Sequence comparisons were conducted using the MegAlign program, version 3.06b (DNASTAR Inc., London, UK). Transmembrane regions were predicted by the TMHMM Server ver. 2.0 (http:// www.cbs.dtu.dk/services/TMHMM/). Prediction of subcellular localization of the deduced amino acids was conducted by using the PSORT (http:// www.psort.nibb.ac.jp/form.html) and TargetP (http://www.cbs.dtu.dk/services/TargetP/) algorithm. Multiple amino acid alignments were performed with CLUSTAL W using default parameters. A phylogenetic tree was constructed using the Neighbor-Joining method and protdist algorithm in the PHYLIP package (version 3.63). The significance level of the neighbor-joining analysis was examined by bootstrap testing with 1000 repeats. The tree was represented by using TREEVIEW (version 6.6) software.

Accession number Sequence data from this article have been deposited at GenBank under accession number DQ532371.

Results and discussion Isolation and sequence analysis of a novel FAD2 gene in soybean Using the degenerate primers P1 and P2, based on highly conserved regions outside of the three histidine boxes characteristic for desaturases (Fig. 1), a distinct cDNA fragment was amplified using RNAs isolated from different soybean tissues as template, with the expected size of about 900 bp after sequencing. We found that the amino acid sequence deduced from this fragment showed high similarity to the known sequences of D-12 FADs from soybean and other plants. To obtain a fulllength cDNA clone, we performed 50 and 30 RACE using gene-specific primers designed from the known cDNA sequence, respectively, and got a

L. Li et al. full-length cDNA (see ‘‘Materials and Methods’’). The clone was 1570 bp long, with an ORF of 1149 bp coding for a polypeptide of 383 amino acids. Alignment of this sequence and the previously reported FAD2-2 (GenBank accession no. L43921; Heppard et al., 1996) showed 95% identity at nucleotide level and 95.3% at amino acid level, differing in 18 amino acids throughout the entire coding region (Fig. 1). To examine whether this sequence is a novel FAD2 gene or merely a new allele of closely related FAD2-2, we utilized FAD2-2 gene specific primers to amplify the same template as above. This led to the isolation of a sequence, identical to FAD2-2 reported by Heppard et al. (1996) (data not shown). Therefore the sequence we isolated using degenerate primers and RACE encoded a novel isoform of FAD2, named as FAD2-3. This sequence probably encoded a microsomelocalized enzyme, as concluded both from the size of the deduced protein (Mt ¼ 43.9 kD), similar to that of other plant microsomal D-12 FADs, and from the lack of any obvious N-terminal transit peptide and also the C-terminal motifs (-KDEL or -KXKXX) that would be required for plastid targeting (Jackson et al., 1990). There was an aromatic amino acid enriched signal at the C-terminus of the protein (-YNNKL), which has been reported to be necessary and sufficient for maintaining localization of the enzymes in the ER (McCartney et al., 2004). Figure 1 showed the amino acid sequence of the protein encoded by FAD2-3 together with the plastidial and microsomal homologs from soybean. All these membrane-bound desaturases contained the three conserved histidine clusters [HX(3 or 4)H, HX(2 or 3)HH and HX(2 or 3)HH] that have been shown essential for desaturase activity-acting as potential ligands for non-heme iron atoms (Shanklin et al., 1994; Shanklin and Cahoon, 1998). A group of enzymes including desaturases, hydrozylases, and epoxygenases found in animals, fungi, plants, and bacteria catalyze the diverse reactions. These proteins probably use a common reactive center, and these histidine-rich motifs are thought to form part of the diiron center where oxygen activation and substrate oxdation occur (Shanklin et al., 1997). A single histidine mutation in three conserved histidine motifs could cause the loss of Spirulina-D6 desaturase activity (Kurdrid et al., 2005). Acyl-lipid and acyl-CoA desaturase are hydrophobic proteins that apparently span the membrane four times, with a portion of the protein, including N- and C-termini and active-site histidine boxes, exposed on the cytosolic side of the membrane (Shanklin et al., 1994). Hydropathy

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analysis of the soybean FAD2 polypeptides was made using the Protein Sequence Analysis software (I.W. Palmer, Gaithersburg, MD, USA), and transmembrane regions were predicted by the TMHMM Server version 2.0 (http://www.cbs.dtu.dk/ services/TMHMM/). There were four tentative membrane-localized domains that correspond to the predicted membrane-spanning domains in desaturase integral membrane protein models (Shanklin et al., 1994) in FAD2 proteins (Fig. 1). The three conserved histidine boxes were located in hydrophilic regions, and according to this topological model, all of them were exposed to the cytoplasmic side (Los and Murata, 1998). To elucidate the phylogenetic relationships of the FAD2, their deduced amino acid sequences were aligned with other homologous D-12 desaturase sequences and an N-J tree was constructed (Fig. 2). As reported previously (Hernandez et al., 2005), these plant D-12 desaturase sequences were classified into three major branches, termed Housekeeping-type FAD2, FAD6 and Seed-type FAD2. FAD2-3 was positioned in a subgroup with FAD2 enzymes that exhibit a housekeeping pattern of expression (Fig. 2). Unlike reports by Hernandez et al. (2005), the position of FAD6 was closer to Housekeeping-type FAD2 than that of the Seed-type FAD2. These observations supported the hypothesis that diverged FAD2 enzymes with the same functionality, but not forming the same clade, may arise independently several times during evolution, and the evolutionary process of Housekeeping-type FAD2, FAD6 and Seed-type FAD2 genes might be different from each other (Dyer et al., 2002; Banilas et al., 2005). In addition, the observed relative high level of divergence within the Housekeeping-type FAD2 group implied that certain genes might have evolved differently, although more data are required to further confirm this observation.

constitutive promoter GAL1 (see ‘‘Materials and methods’’) and expressed in yeast to confirm its enzymatic activity. Using the empty vector pYES2 as negative control and the pYES2-FAD2-2 as positive control, these different constructs were transformed into the INVSc1 strain of S. cerevisiae and expressed at 30 1C until stationary phase. From Fig. 3, we may see that yeast cells transformed with the empty vector pYES2 showed the typical yeast fatty acids (16:0, 16:1D9, 18:0 and 18:1D9) and the expression of FAD2-2 and FAD2-3 resulted in an additional peak corresponding to 18:2D9,12. In addition, the oleate (18:1) peak in the transformed cells was noticeably smaller than the corresponding oleate peak in the negative control cells, indicating that the conversion of oleate into linoleate in the yeast cells containing the FAD2-2 and FAD2-3 genes (Fig. 3). Thus, the newly isolated soybean FAD2-3 gene has been functionally identified since it encodes a microsomal oleate desaturase isoform that catalyzes the desaturation of the endogenous oleate to linoleate. It has previously been shown that plant desaturase can heterologously expressed in Escherichia coli, but the enzyme cannot function in E. coli unless the reaction was carried out in the presence of exogenous cofactors, like ferredoxin (Cahoon et al., 1996). Yeast is an ideal background for the study of plant oleate desaturase genes because it provides a suitable membrane environment (the ER) and the requisite electron donor (cytochrome b5), and lacks enzyme activities capable of synthesizing PUFAs. The fact that FAD2-2 and FAD2-3 successfully expressed and functioned in S. cerevisiae further supported the hypotheses that plant desaturase is able to use an electron donor in yeast cells for desaturation reaction (Sperling et al., 2003).

Functional expression of the soybean FAD2-3 gene in S. cerevisiae

Tissue-specific and developmentally regulated expression of soybean FAD2-3 gene

Yeast is an excellent model system for analyzing the function of plant lipid-modifying enzymes, because it lacks the polyunsaturated or exotic fatty acids typically found in plants. Yeast cells have been used successfully for functional expression of several plant microsomal desaturases localized in the ER, such as the Arabidopsis FAD2 enzyme (Covello and Reed, 1996), the tung FAD2 gene (Dyer et al., 2002), the olive FAD2 genes (Hernandez et al., 2005). To confirm the functional identity of the soybean FAD2-3 gene, we cloned its ORF into the expression vector pYES2 (Invitrogen) behind the

To investigate the physiological role of the FAD23 gene, we examined the transcript level by semiquantitative RT-PCR using FAD2-3 gene-specific primers. Total RNA was isolated from developing seeds at different stages (11, 14, 19, 20, 22, 24, 27 and 32 DAF), leaves, stems, roots, and embryos (22 DAF), cotyledons (22 DAF), seed coats (22 DAF). The expression analysis showed that FAD2-3 gene was expressed in developing seeds and all tissues examined, and appeared to be constant during seed development (Fig. 4), suggesting that FAD2-3 gene

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Figure 2. Phylogenetic relationships between deduced amino acid sequences from soybean FAD2 and other plant microsomal (FAD2) or plastidial (FAD6) oleate desaturases. Position of the soybean FAD2-3 is framed. The enzymes and GenBank accession numbers used for the analysis are: Arabidopsis thaliana (AtFAD2, L26296; AtFAD6, U09503), Arachis duranensis (AdFAD2, AF272951), Arachis hypogaea (AhFAD2A, AF030319; AhFAD2B, AF272950), Arachis ipaensis (AiFAD2, AF272952), Borago officinalis (BoFAD2, AF074324), Brassica carinata (BcFAD2, AF124360), Brassica juncea (BjFAD2, X91139), Brassica napus (BnFAD2, AF243045; BnFAD6, L29214), Brassica rapa (BrFAD2, AJ459107), Calendula officinalis (CoFAD2, AF343065), Crepis palestina (CpaFAD2, Y16284), Cucurbita pepo (CpeFAD2, AY525163), Euphorbia lagascae (ElFAD2, AY486148), Glycine max (GmFAD2-1A, L43920; GmFAD2-1B, AB188251; GmFAD2-2, L43921; GmFAD6, L29215), Gossypium hirsutum (GhFAD2-1, X97016; GhFAD2-2, Y10112; GhFAD2-3, AF331163), Helianthus annuus (HaFAD2-1, AF251842; HaFAD2-2, AF251843; HaFAD2-3, AF251844), Persea americana (PamFAD2, AY057406), Petroselinum crispum (PcFAD2, U86072), Punica granatum (PgFAD2, AJ437139), Sesamum indicum (SiFAD2, AF192486), Solanum commersonii (ScFAD2, X92847), Spinacia oleracea (SoFAD2, AB094415; SoFAD6, X78311), Vernicia fordii (VfFAD2, AF525535), Vernonia galamensis (VgFAD2-2, AF188264). The tree was constructed by using the Neighbor-Joining algorithm. Numbers on the nodes indicate bootstrap values after 1000 replicates.

was constitutively expressed in both vegetative tissues and developing seeds. Combined with the previously reported results (Heppard et al., 1996; Tang et al., 2005), there were four different FAD2 encoding microsomal oleate desatrases in soybean. These seed-specifically expressed FAD2-1A and FAD2-1B genes were most likely having major roles in converting oleic acid to linoleic acid during storage lipid biosynthesis in soybean seed development. The FAD2-2 and FAD2-3 gene-encoded oleate desaturases appeared

to be responsible for production of PUFAs within membrane lipids in both vegetative tissues and developing seeds. Taking into account of the total number of FAD2 genes involved, the differential stabilities of FAD2-1A versus FAD2-1B, and the potential of control via phosphorylation (Tang et al., 2005), it is likely that the regulation of 18:1 desaturation during triacylglycerol synthesis in soybean is complex. Thus it seems reasonable that six independent quantitative trait loci (QTL) have been mapped governing seed 18:1 content among

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different soybean genotypes and/or environments (http://soybase.agron.iastate.edu). The presence of two seed-specific FAD2 genes was previously reported for peanut (Jung et al., 2000), and one seed-specific and two constitutive FAD2 genes were also previously reported for cotton (Liu et al., 1999; Pirtle et al., 2001) and sunflower (MartinezRivas et al., 2001). In peanut, both two seedspecific FAD2 genes, like soybean seed-specific FAD2, were expressed with the highest levels in developing seeds (Jung et al., 2000). The sunflower constitutive FAD2 genes were weakly and uniformly expressed in all tissues, except for cotyledons after 2 days of germination and for roots, where the expression of one FAD2 was higher (Martinez-Rivas et al., 2001). Whereas the transcript levels for the

soybean FAD2-3 genes were not significant discrepancies in all tissues examined (Fig. 4). Soybean expression sequenced tags (ESTs) database (Gene Index) contains over 330,000 sequences from 84 cDNA libraries (http://www.tigr.org/tdb/ tgi/plant.shtm), these resources offer the possibility to identify the gene expression profile in silico. Eighty-four cDNA libraries were classified and grouped into eight virtual synthetic libraries based on the organ used for construction of each cDNA library (see ‘‘Materials and methods’’). The ESTs of FAD2-3 gene were assigned into each synthetic library. A method developed by Audic and Claverie (1997) was used to calculate the probability of differential expression of a given gene between two libraries, taking into account the number of ESTs in each synthetic library and the size of the two libraries. The results of in silico ESTs-derived expression profile analysis were consistent with the results revealed by semi-quantitative RT-PCR (Table 1, Fig. 4).

Expression of soybean FAD2-3 in response to low temperature

Figure 3. Gas chromatographic analysis of fatty acid methyl esters extracted from yeast transformants using flame ionization detector (FID). Yeast S. cerevisiae strain INVSc1 cells (Invitrogen) transformed with pYES2 (A), pYES2-FAD2-3 (B) and pYES2-FAD2-2 (C) were grown in synthetic complete minus uracil (SCU) medium containing 2% galactose and 2% raffinose at 30 1C until stationary phase.

Table 1.

Seed Root Leaf Flower Seedling Stem

The composition of saturated and unsaturated fatty acids of both membrane and storage lipids varies depending on environmental temperature. The level of unsaturation of membrane fatty acids, as well as seed storage lipids, has been shown inversely correlated with growth temperature (Rennie and Tanner, 1989; Heppard et al., 1996). Heppard et al. (1996) reported that the elevated PUFA levels in leaves grown at low temperature were not due to enhanced expression of FAD2-2 in soybean. To test whether the increase of PUFA levels in leaf by low temperature is related to enhanced FAD2-3 expression, we determined the level of transcripts by semi-quantitative RT-PCR analysis. The transcript level of FAD2-3 gene was relatively constant in leaves at different growth temperatures, and the low grown temperature did

Probabilities of differential expression between organs for FAD2-3 Seed (30)

Root (15)

Leaf (11)

Flower (3)

Seedling (12)

Stem (2)

— 0.4–0.5 0.5–0.6 0.97–0.98 0.7–0.8 0.1–0.2

0.4–0.5 — 0.1–0.2 0.98–0.99 0.4–0.5 0.3–0.4

0.5–0.6 0.1–0.2 — 0.8–0.9 0.3–0.4 0.3–0.4

0.97–0.98 0.98–0.99 0.8–0.9 — 0.7–0.8 0.8–0.9

0.7–0.8 0.4–0.5 0.3–0.4 0.7–0.8 — 0.5–0.6

0.1–0.2 0.3–0.4 0.3–0.4 0.8–0.9 0.5–0.6 —

Probabilities are indicated in bold when they are 40.95 and correspond to an up-regulation in the organs also indicated in bold. Nos. of ESTs is indicated between brackets.

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Figure 4. Expression profile of FAD2-3 in developing soybean seeds and other tissues by semi-quantitative RTPCR analysis. (A) Expression analysis in developing seeds (11, 14, 19, 20, 22, 24, 27 and 32 DAF). (B) Spatial expression analysis in embryo (em), cotyledon (co), seed coat (sc), leaf (l), stem (s) and root (r). Embryo, cotyledon and seed coat were derived from the 22 DAF seeds. b-Actin was used as a reference gene.

L. Li et al. with 18:3 content exhibiting gradual increase and 18:2 content exhibiting gradual decrease. Both of 18:3 and 18:2 remained relatively constant after 1-day time point, until the final time point 1 week. But no obvious change of 18:1 and 18:0 content was observed at all the time points (Fig. 6A). The transcript levels of FAD2-2 and FAD2-3 genes were relatively constant in the time-course analysis of low-temperature treatment (Fig. 6B), although low temperature did decrease significantly the level of 18:2. These results indicated that low-temperature treatment appeared to cause an increase of 18:3 content due to the flux from 18:2 fatty acid. The fatty acid composition is greatly influenced by growth temperature for both prokaryotic and eukaryotic organisms. The unsaturated fatty acids have lower melting points than their saturated counterparts, and content of PUFAs increases to confer greater membrane fluidity, which may maintain membrane function under lower growth temperature conditions (Heppard et al., 1996). There may be two different mechanisms to

Figure 5. Growth temperature effects on expression of FAD2-2 and FAD2-3 in soybean leaves. Total RNA was extracted from young leaves grown at 32/28, 28/22, 18/12 and 12/8 1C, respectively. b-Actin was used as a reference gene.

not result in enhanced expression FAD2-3 (Fig. 5). Interestingly, the expression of FAD2-2 was lower than that of FAD2-3 at different growth temperatures, and the transcript of FAD2-2 was not detected at high growth temperature (32/28 1C). These results further confirmed the hypothesis that the increase level of PUFAs by low temperature is likely the result of translation and posttranslational regulation, such as altered desaturase enzyme activity (Cheesbrough, 1989), rather than transcriptionally induced or enhanced expression of the characterized FAD2 genes in soybean. Based on the substantial change of soybean membrane fatty acids detected at different grown temperatures (Heppard et al., 1996), we expected to determine the alternation of the membrane fatty acids in response to cold stress and the relationship between the alterations and gene expression. Soybean seedlings were grown at 28 1C for 12 days and then shifted to 8 1C. By GC analysis, the fatty acid composition was then evaluated in leaves sampled at various times after the shift to low temperature. No significant change was detected in the fatty acid profile 4 h after the shift to 8 1C. The earliest time point reflecting an alteration in response to low temperature occurred at 8 h,

Figure 6. Time-course analysis of the leaf membrane fatty acid composition and the transcript levels of FAD2-2 and FAD2-3 in response to low-temperature treatment. Plants were grown for 12 d at 28 1C before being shifted to low temperature (8 1C). (A) Fatty acid profile of leaves was determined by gas chromatographic analysis at the designated times over 1 week after the shift to low temperature. The values are averages from two independent assays for each time point. (B) Semi-quantitative RT-PCR analysis of transcript levels for FAD2-2 and FAD2-3 in response to low-temperature treatment. b-Actin was used as a reference gene.

ARTICLE IN PRESS A novel oleate desaturase gene in soybean regulate the fatty acid composition of soybean leaf lipids in response to low grown temperature and the shift to low temperature, respectively. The low growth temperature caused to an increase in the 18:3 content but maintaining the relatively constant level of 18:2, which may exist a mechanism to retain a certain level of 18:2 by balancing the flux through 18:1 and 18:2 fatty acids (Heppard et al., 1996). The increase of the soybean leaf 18:3 content was observed after the shift to low temperature (8 1C) from the high growth temperature (28 1C), however, the proportion of 18:2 showed a decline trend (Fig. 6A), and the expression of soybean FAD2-2 and FAD2-3 was not influenced (Fig. 6B). Another mechanism for the shift to low temperature may be due to D-15 FAD converting 18:2 to 18:3 to maintain membrane function.

Acknowledgments We thank Ms. Hao Cheng for her experimental assistance and Dr. Qingchang Meng for his helpful discussion. This work was supported in part by National 973 Projects (Nos. 2004CB117206 and 2002CB111304), National Natural Science Foundation of China (No. 30490250), National 863 Project (No. 2002AA211052), and an award grant for Outstanding Scholars from the Ministry of Education of China.

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