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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Effect of Temperature on Microsomal Omega-3 Linoleate Desaturase Gene Expression and Linolenic Acid Content in Developing Soybean Seeds

^a Microbiology Dep., North Carolina State Univ., Raleigh, NC 27695
^b USDA-ARS, Soybean and Nitrogen Fixation Unit and Plant Pathology Dep., North Carolina State Univ., Raleigh, NC 27695

^* Corresponding author (greg_upchurch{at}ncsu.edu).

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The mechanism of temperature adaptation in plants, including the formation of polyunsaturates in seed storage lipids, most likely involves transcriptional as well as post-translational regulation of fatty acid desaturase activity. The present investigation was conducted to measure changes in the transcript accumulation among the three members of the soybean [Glycine max (L.) Merr.] microsomal omega-3 fatty acid desaturase gene family in response to altered growth temperature during seed development. Microsomal omega-3 fatty acid desaturases catalyze the insertion of a third double bond into linoleic (18:2^{9, 12}) acid to produce linolenic (18:3^{9, 12, 15}) acid. At 35 d after flowering, transcript accumulation (normalized for soybean actin) of GmFAD3A decreased by 5- to 15-fold, GmFAD3B by 2- to 9-fold, and GmFAD3C by 2- to 3-fold in seeds that developed in a warm (day/night [D/N] = 30/26°C) versus a normal (D/N = 26/22°C) or a cool (D/N = 22/18°C) environment. At this stage of seed development, decreased omega-3 desaturase gene expression levels were positively associated with reductions of 39 to 50% in the linolenic acid content of seeds of three soybean varieties examined. Thus, transcriptional regulation of the microsomal omega-3 fatty acid desaturase gene family likely accounts, at least in part, for the reduced linolenic acid levels in soybean seeds grown at elevated temperature.

Abbreviations: bp, base pair • DAF, days after flowering • D/N, day/night • FAME, fatty acid methyl ester • GmFAD3, Glycine max omega-3 fatty acid desaturase • PCR, polymerase chain reaction • RT-PCR, reverse transcription–polymerase chain reaction

Effect of Temperature on Microsomal Omega-3 Linoleate Desaturase Gene Expression and Linolenic Acid Content in Developing Soybean Seeds

^a Microbiology Dep., North Carolina State Univ., Raleigh, NC 27695
^b USDA-ARS, Soybean and Nitrogen Fixation Unit and Plant Pathology Dep., North Carolina State Univ., Raleigh, NC 27695

^* Corresponding author (greg_upchurch{at}ncsu.edu).

The mechanism of temperature adaptation in plants, including the formation of polyunsaturates in seed storage lipids, most likely involves transcriptional as well as post-translational regulation of fatty acid desaturase activity. The present investigation was conducted to measure changes in the transcript accumulation among the three members of the soybean [Glycine max (L.) Merr.] microsomal omega-3 fatty acid desaturase gene family in response to altered growth temperature during seed development. Microsomal omega-3 fatty acid desaturases catalyze the insertion of a third double bond into linoleic (18:2^{9, 12}) acid to produce linolenic (18:3^{9, 12, 15}) acid. At 35 d after flowering, transcript accumulation (normalized for soybean actin) of GmFAD3A decreased by 5- to 15-fold, GmFAD3B by 2- to 9-fold, and GmFAD3C by 2- to 3-fold in seeds that developed in a warm (day/night [D/N] = 30/26°C) versus a normal (D/N = 26/22°C) or a cool (D/N = 22/18°C) environment. At this stage of seed development, decreased omega-3 desaturase gene expression levels were positively associated with reductions of 39 to 50% in the linolenic acid content of seeds of three soybean varieties examined. Thus, transcriptional regulation of the microsomal omega-3 fatty acid desaturase gene family likely accounts, at least in part, for the reduced linolenic acid levels in soybean seeds grown at elevated temperature.

Abbreviations: bp, base pair • DAF, days after flowering • D/N, day/night • FAME, fatty acid methyl ester • GmFAD3, Glycine max omega-3 fatty acid desaturase • PCR, polymerase chain reaction • RT-PCR, reverse transcription–polymerase chain reaction

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE -LINOLENIC ACID CONTENT of oil from wild-type soybean [Glycine max (L.) Merr.] varieties is typically 6 to 10%. Since this level of linolenic acid is too high to ensure oxidatively stable oil for current market demands, research has focused on enhancing the selection of soybean varieties with reduced-seed -linolenic acid. Soybean lines have been developed through mutagenesis breeding with mutations at the fan locus, and these lines produce oil containing lower -linolenic acid (Wilcox and Cavins, 1985; Rennie and Tanner, 1991; Fehr et al., 1992; Rahman et al., 1996, 1998; Ross et al., 2000). Fan is the major locus controlling the linolenic acid level in soybean seeds.

Omega-3 fatty acid desaturases catalyze the insertion of a third double bond into linoleic acid (18:2^{9, 12}) to produce linolenic acid (18:3^{9, 12, 15}). Bilyeu et al. (2003) identified and characterized a family of Glycine max microsomal omega-3 fatty acid desaturase (GmFAD3) genes. Using database homology searches with an Arabidopsis omega-3 fatty acid desaturase query sequence, they found three soybean microsomal omega-3 fatty acid desaturase genes in cultivar Pana, which they designated as GmFAD3A, GmFAD3B, and GmFAD3C, that contribute to seed linolenic acid content. Quantitative real-time polymerase chain reaction (PCR) measurements of relative gene expression indicated that one of the genes, GmFAD3A, was predominately expressed in developing seeds. The low -linolenic acid line A5 (fan fan) was found to express two of the desaturase genes but lacked the sequence of GmFAD3A; thus, they assigned the Fan locus to GmFAD3A.

Anai et al. (2005) screened a cDNA library of soybean cultivar Bay with a soybean FAD3 probe and obtained four seed cDNAs, which they designated GmFAD3-1a, GmFAD3-1b, GmFAD3-2a, and GmFAD3-2b, that encoded microsomal omega-3 fatty-acid desaturases. Two of these FAD3 genes were identified that contributed to the -linolenic acid content in developing soybean seed. GmFAD3 genes from three X-ray induced soybean mutants were sequenced and their expression levels semiquantitatively determined. Oil from the seeds of cultivar Bay contains 6.2% linolenic acid. They found that GmFAD3-1b (equivalent to GmFAD3A) was deleted from the genome of the low seed linolenic acid (3.0%) mutant J18, that the coding region of GmFAD3-1b from mutant M5 (3.3% linolenic acid) contained a 19 base pair deletion, and that the coding region of GmFAD3-1a (equivalent to GmFAD3B) in mutant M24 (4.5% linolenic acid) contained a single base pair deletion. The FAD3 mutant gene products from M5 and M24 had complete loss of enzymatic activity when expressed heterologously in yeast. The contribution of the GmFAD3-1b gene product to -linolenic acid accumulation in soybean seed was found to be greater that that of the GmFAD3-1a gene product. They designated the mutant allele in J18 and M5 (GmFAD3-1b, equivalent to GmFAD3A) as fan and the mutant allele in M24 (GmFAD3-1a, equivalent to GmFAD3B) as fanx^a.

Bilyeu et al. (2005) characterized the GmFAD3 genes they had previously identified from low linolenic acid soybean lines to associate the alleles with that trait. From their studies, they found that two of the FAD3 genes contained mutations. Soybean line CX1512-44 and the homologous, derived F₆ line 2721 both contained a frameshift mutation in GmFAD3A and a single nucleotide mutation in GmFAD3C, rendering the omega-3 desaturases encoded by these two genes inactive. A detection method for single nucleotide polymorphisms in GmFAD3A and GmFAD3C using cleaved amplified polymorphic sequence assays was developed to characterize omega-3 desaturase soybean genotypes. Genotypes containing homozygous mutant alleles of both genes showed an additive but unequal reduction of more than 67% of the linolenic acid present in wild-type seed. In lines homozygous for mutant GmFAD3A (aa) and mutant GmFAD3C (cc), the reduction of linolenic acid was 46 mg g^–1 oil for aa, and 11 mg g^–1 oil for cc, compared with a wild-type soybean variety (AACC). Their results demonstrated that the mutant omega-3 desaturase genotypes could be identified with the mutation-specific molecular markers in the F₂ generation, and that the low linolenic acid trait was stably inherited in subsequent generations. The recent work of Beuselinck et al. (2006) demonstrated that accurate phenotypic selections in soybean for low seed -linolenic acid content (<35 mg g^–1 oil) could be made using molecular markers specific for mutations in the two fatty acid desaturase genes GmFAD3A and GmFAD3C, provided that diligence is exercised to eliminate errors in sampling and fatty acid analysis.

The above studies show that -linolenic acid content in soybean seeds is heritable and significantly the result of omega-3 fatty acid desaturase gene action. Other research has shown that environmental temperature influences both oil content and fatty acid composition in developing seed. Temperature affects the total oil content of soybeans as evidenced by the positive correlation between maximal temperature and oil percentage (Wilson, 2004). Temperature has also been consistently shown to be a major factor affecting seed fatty acid composition, especially for the unsaturated fatty acids. An investigation using controlled growth chambers found that the fatty acid composition of developing seed was strongly affected by temperature: linoleic and linolenic acids decreased whereas oleic acid increased as temperature increased (Wolf et al., 1982). Field studies by Hou et al. (2006) demonstrated that linoleic and linolenic acid levels of normal soybean varieties were particularly vulnerable to differences in environmental temperature.

Advances have been made in elucidating the mechanisms of temperature regulation of fatty acid composition in soybean. Growth temperature was shown to affect linoleoyl desaturase enzyme activity in soybean seeds from pods cultured in vitro (Cheesbrough, 1989). Linoleoyl desaturase enzyme activity decreased 98% in assays conducted at 25°C compared with 20°C. Tang et al. (2005) demonstrated that two post-translational regulatory mechanisms, namely, elevated temperature-induced instability of the FAD2-1A isoform and phosphorylation of the FAD2-1 proteins likely play important roles in modulating activity of the FAD2-1 enzymes. Thomas et al. (2003) demonstrated that linolenic acid levels declined with increasing temperature as did the transcript accumulation of a gene involved in normal seed development. We have shown that delta-9 stearoyl-acyl carrier protein desaturase and omega-6 oleate desaturase gene transcripts are significantly decreased in seeds that develop in a warm (day/night [D/N] = 30/26°C) versus cool (D/N = 22/18°C) temperatures during pod fill (Byfield and Upchurch, 2007). Decreased omega-6 oleate desaturase transcript accumulation at the warm temperature was positively associated with significantly increased oleic and decreased linoleic acid content in soybean seeds. Decreased delta-9 stearoyl-acyl carrier protein desaturase transcript accumulation at the warm temperature was positively associated with a significantly increased level of stearic acid in the high stearate mutant line A6.

The present investigation was conducted to determine to what extent omega-3 fatty acid desaturase transcript accumulation changes in developing soybean seeds in response to changes in growth temperature and how these changes are associated with -linolenic acid content in seeds. Here we show that transcript accumulation of GmFAD3A, GmFAD3B, and GmFAD3C decreases during seed development at a warm (D/N = 30/26°C) versus a normal (D/N = 26/22°C) or cool (D/N = 22/18°C) temperature, and that decreased omega-3 desaturase gene expression is positively associated with decreased linolenic acid content in seed.

	MATERIALS AND METHODS

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Plant Varieties and Growth Conditions
Three soybean varieties were chosen on the basis of seed linoleic and linolenic acid content at maturity. Oil from field-grown maturity group V determinate soybean cultivar Dare (Brim, 1966) contains 58% linoleic acid and 8% linolenic acid, oil from field-grown maturity group VI determinate soybean line N99-3170 (Wilson et al., 2001) has a relatively high linoleic acid content of 64% and reduced linolenic acid content of 4%, and oil from field-grown maturity group IV, indeterminate mid-oleic line N01-3544 (Burton et al., 2005), has reduced linoleic (33%) and reduced linolenic (3%) content. Seeds were supplied by J.W. Burton, USDA-ARS, Raleigh, NC. Plants were grown in semicontrolled chambers at the Southeastern Plant Environment Laboratory (Phytotron) at North Carolina State University, Raleigh, NC. Pregerminated seeds were planted in 25-cm pots containing standard mix (1/3 peat-lite and 2/3 gravel), and five plants of each variety were grown in a random arrangement in each of three chambers. Chambers were initially maintained at a D/N temperature of 26/22°C (normal) with 14 h of incandescent light and 10 h of darkness, with a 1-h light interruption of the dark to synchronize flowering. Plants were watered twice daily with deionized water, and nutrient solution was supplied thrice weekly. On Day 15, the light interruption was stopped and chambers were reset to D/N = 12/12 h to induce flowering. On Day 36, the air temperatures were changed in two chambers—one to 22/18°C (cool), D/N = 12/12, and the other to 30/26°C (warm), D/N = 12/12—while the third chamber remained at 26/22°C (normal) with D/N = 12/12. These temperatures were maintained through the end of the experiment. Seeds were harvested at four developmental stages between R5 (beginning of seed set) and R6 (mature bean), coinciding with 18, 23, 28, and 35 d after flowering (DAF) from three plants of each variety in each chamber and quickly flash frozen in liquid nitrogen and stored at –80°C.

RNA Isolation and Real-Time Reverse Transcription–Polymerase Chain Reaction
RNA was isolated from three seed samples (each from a different plant) of each soybean variety per temperature treatment for each of the four developmental stages. The RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and the manufacturer's protocol were used to isolate total RNA from 100 mg of frozen powdered tissue per sample. RNA samples were DNase treated with DNA-free (Ambion, Houston, TX) according to the manufacturer's directions. RNA concentrations were determined using a DU-640 spectrophotometer (Beckman Coulter, Fullerton, CA) at absorbance 260 nm. Aliquots of RNA (free of genomic DNA) were diluted to 50 ng µL^–1 in RNase-free water and stored at –80°C until use. To verify RNA integrity, 500 ng of total RNA of each sample was examined on a 1% agarose gel following electrophoresis and staining with ethidium bromide.

Real-time reverse transcription–polymerase chain reaction (RT- PCR) was performed with the iCycler (Bio-Rad, Hercules, CA) using the SYBR Green RT-PCR kit (Qiagen, Valencia, CA) to quantify GmFAD3A, GmFAD3B, GmFAD3C, and soybean actin transcripts. FAD3 gene-specific primers GmFAD3A (5'-AGCGACACAAGCAGCAAAAT-3' and 5'-GTCTCGGTGCGAGTGAAGGT-3'), GmFAD3B (5'-TCCACCCAGTGAGAGAAAA-3' and 5'-AGCACTAGAAGTGGACTAGTTATGAAT-3'), and GmFAD3C (5'-GCTGGGAGAAGAACACATTGAG-3' and 5'-CCCAAAACATTGTGCCTTG-3') were those designed by Bilyeu et al. (2003). Primers used for the housekeeping gene soybean actin (5'-GAGCTATGAATTGCCTGATGG-3' and 5'-CGTTTCATGAATTCCAGTAGC-3') were designed by Byfield et al. (2006) based on the GenBank accession number U60500 (Moniz de Sa and Drouin, 1996). Polymerase chain reactions with the FAD3 and actin primers produced the following amplicons: GmFAD3A, 183 base pairs (bp); GmFAD3B, 104 bp; GmFAD3C, 143 bp; and actin, 188 bp. A typical reaction, done in duplicate, contained 12.5 µL of 2X SYBR Green PCR master mix, 250 nM each primer, 0.25 µL RT mix, 250 ng total RNA, and nuclease-free water to 25 µL. Conditions for reverse transcription and amplification were 50°C for 30 min followed by 95°C for 15 min and then 40 cycles of 15 s at 94°C, 30 s at the annealing temperature, and 30 s at 72°C. Annealing temperatures were 60°C for GmFAD3A, GmFAD3C, and actin, and 56.5°C for GmFAD3B. A melt curve analysis over a 10°C temperature gradient at 0.05°C s^–1 from 78° to 88°C was done after amplification to verify that a single product was produced in each reaction.

The transcript abundance of the GmFAD3 genes and actin were mathematically determined by comparison of individual PCR cycle threshold values with a standard curve generated from serial dilutions of the respective authentic transcript fragment. Control reactions with RNA template but without reverse transcriptase produced no product. Copy number, initially determined per microgram of total RNA, was normalized to the soybean actin gene expression level.

Analysis of Fatty Acid Content
Seed samples used for RNA extraction were also the source material for fatty acid analysis. Fatty acid methyl esters (FAMEs) of the seed samples were prepared using acid methanolysis. Powdered seed tissue was heated to and held at 85°C for 90 min. in a 5% HCl–95% methanol solution. FAME was partitioned 2x into hexane and transferred to 2-ml vials for analysis. The FAMEs were separated by gas chromatography using an HP 6890 GC (Agilent Technologies, Inc., Wilmington, DE) equipped with a DB-23 30 m x 0.53 mm column (Agilent Technologies, Inc., Wilmington, DE). Operating conditions were 1-µL injection volume, a 20:1 split ratio, and He carrier gas flow of 6 mL min^–1. Temperatures were 250°C, 200°C, and 275°C for the injector, oven, and flame ionization detector, respectively. Peak areas of the chromatograms were analyzed using HP ChemStation software and fatty acid contents, as per cents, were calculated as mg fatty acid g^–1 oil.

Statistical Analysis
The accumulations of GmFAD3 and actin transcripts and the accumulation of oleic, linoleic, and linolenic acids in seed across temperatures were statistically analyzed for significance (ANOVA) online at http://www.physics.csbsju.edu/stats/anova.html.

	RESULTS

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Effect of Temperature on Linolenic Acid Content in Developing Seed
The oleic, linoleic, and linolenic acid contents in seeds of the three soybean varieties, at 28 and 35 DAF, are shown in Table 1 and shown graphically for 35 DAF data in Fig. 1 . Representative gas chromatography fatty acid profiles for seed samples of the three soybean varieties produced under the three air temperature regimes are shown in Fig. 2 .

View larger version (29K): [in this window] [in a new window]   Figure 1. Effect of temperature (day/night) on mean oleic, linoleic, and linolenic acid content (mg g–1 oil) of developing soybean seeds of Dare, N99-3170, and N01-3544 at 35 d after flowering (DAF). Values represent mean ± SE (p < 0.05).

View larger version (32K): [in this window] [in a new window]   Figure 2. Selected gas chromatography traces showing the effect of temperature (day/night [D/N]) on seed fatty acid composition at 35 d after flowering: left to right, cool (D/N = 22/18°C); normal (D/N = 26/22°C); warm (D/N = 30/26°C). (A, B, C) Soybean cultivar Dare; (D, E, F) soybean variety N99-3170; (G, H, I) soybean variety N01-3544.

Effect of Temperature on Omega-3 Desaturase Gene Expression
The transcript levels (copy number per µg total RNA, normalized to actin) at 28 and 35 DAF of the three soybean FAD3 genes are shown in Table 2 , and graphically for 35 DAF in Fig. 3 . The data reveal that temperature affects the pattern of transcript accumulation in seeds in a statistically significant (p < 0.05) and similar manner for the three omega-3 desaturase genes. GmFAD3A, B, and C mean transcript accumulations declined dramatically in seeds that developed in the warm temperature environment compared with those that developed in the cool temperature environment. Steep (GmFAD3A) to intermediate (GmFAD3B and C) declines in transcript abundance of the FAD3 genes were detected in seed that developed in the normal air temperature (D/N = 26°C) (Table 2, Fig. 3). Across the three varieties at 35 DAF, GmFAD3A had decreased by 5- to 15-fold, GmFAD3B by 2- to 9-fold, and GmFAD3C by 2- to 3-fold in seeds that developed in the warm versus the cool environment. We found, as Bilyeu et al. (2003) first reported, that GmFAD3A was the most abundant FAD3 transcript but, in our study, only in seeds that developed in the cool (D/N = 22/18°C) temperature at 35 DAF. At 28 DAF in all three varieties, GmFAD3B transcript was more abundant than GmFAD3A or GmFAD3C in seeds taken from the cool environment. Similar trends were observed for the effects of temperature on omega-3 desaturase transcript accumulation at 18 and 23 DAF, but the data (not shown) were not always statistically significant (p > 0.05).

View larger version (19K): [in this window] [in a new window]   Figure 3. Effect of temperature (day/night) on the mean expression of the GmFAD3 genes in developing soybean seeds of varieties Dare, N99-3170, and N01-3544 at 35 d after flowering. Values are normalized to actin and represent mean ± SE (p < 0.05).

	DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Environmental temperature interacts with plant genotype as well. Previous studies (Wolf et al., 1982; Neidleman, 1987; Rennie and Tanner, 1989; Thompson, 1993: Williams et al., 1996; Wilson et al., 2001; Rajcan et al., 2005; Hou et al., 2006) have consistently reported a strong association between higher temperatures during seed development and low linoleic, low linolenic, and high oleic acid relative amounts in soybean varieties with wild-type gene alleles for these traits. Results from temperature manipulation experiments with the three soybean varieties reported here also reflect this association (Table 1, Fig. 1). The response of developing seeds to elevated temperature adaptation produces shifts in oleic, linoleic, and linolenic acid composition and likely involves transcriptional (Byfield and Upchurch, 2007) as well as post-translational regulation (Tang et al., 2005) of microsomal desaturase activity. A recent study identified two omega-3 desaturase genes that control linolenic acid levels in flax (Linum usitatissimum) seed (Vrinten et al., 2005). The level of both LuFAD3A and LuFAD3B transcript was greatly reduced in the low-linolenic acid mutant flax line ‘Solin’. This study concluded that the reduction of -linolenic acid in the mutant reflected both reduced LuFAD3 expression and reduced specific activity of each gene product. LuFAD3A and LuFAD3B transcript accumulation was found to be highest at the cold growth temperature. We found that the three soybean omega-3 fatty acid desaturase genes were most highly expressed in seeds of soybeans grown at the cool temperature during pod fill (Table 2, Fig. 3). Across varieties at 35 DAF, GmFAD3A increased by 5- to 15-fold, GmFAD3B by 2- to 9-fold, and GmFAD3C by 2- to 3-fold in seeds that developed in the cold versus the warm environment. At the cool temperature, GmFAD3A was most highly expressed, followed by GmFAD3B and then GmFAD3C. Thus, our findings show that cool temperature elevates the levels of GmFAD3A, FAD3B, and FAD3C expression in soybean seeds and also confirms the results of Bilyeu et al. (2003), showing GmFAD3A to be more highly expressed than GmFAD3B and GmFAD3C in soybean seed. Transcript accumulation of the omega-3 desaturase genes was much lower at the normal and warm temperatures, and differences in expression among the GmFAD3 genes between these two temperatures were slight. No significant varietal differences were detected for omega-3 desaturase gene expression at the normal and warm temperatures. At 28 and 35 DAF at the cool temperature, a significant varietal difference for the expression of GmFAD3A was observed, with Dare showing the highest expression level. Significant varietal differences were also found for GmFAD3B at the cold temperature. At this temperature, 28 DAF, Dare and the mid-oleic acid line N01-3544 had significantly higher GmFAD3B transcript accumulation compared with GmFAD3B measured in line N99-3170.

We have shown that during seed development (R5–R6), linolenic (as well as oleic and linoleic acid) content in three soybean varieties with normal fan alleles can be controlled by growth temperature. At the cool temperature, Dare seeds contained 13%, N99-3170 seed contained 8.6%, and N01-3544 seed contained 4.8% linolenic acid. In contrast, when seeds of these varieties developed in a warm temperature (D/N = 30/26°C) environment, linolenic acid content decreased to 7.9% in Dare, to 4.1% in N99-3170, and to 2.4% in N01-3544. Comparing these reduced seed linolenic acid levels achieved by warm temperature manipulation with linolenic acid levels reported for soybean omega-3 desaturase mutants grown in the field is instructive. That this type of comparison is possible is suggested by the finding that soybean genotypes with mutant fan alleles conditioning reduced linolenic acid have been found to exhibit this phenotype stably under a wide variety of temperature environments, as evidenced by much lower b values than the stability values for wild-type fan alleles (Primomo et al., 2002). Mutants of cultivar Bay (6.2% linolenic acid), J18, M5, and M24, contained 3.0%, 3.3%, and 4.5% linolenic acid, respectively, and the soybean natural mutant lines CX1512-44 and 2721 contained 2.8% linolenic acid. Thus, the reductions we measured in oil linolenic acid content caused by growth in a warm temperature environment are, by comparison, substantial. Concomitant with the decreased levels of linolenic acid we measured in the oil of soybean seeds grown in the warm environment were significantly decreased transcript accumulations of the three soybean omega-3 desaturase genes.

	ACKNOWLEDGMENTS

 
We thank William P. Novitzky for the fatty acid analysis and the staff of the Southeastern Plant Environmental Laboratory at North Carolina State University for growth chamber space. Mac Rich provided excellent technical assistance. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Received for publication May 8, 2007.

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