**4. Soybean omega-3 linoleate fatty acid desaturases**

The membrane lipids of higher plants including soybean are characterized by a high proportion of polyunsaturated fatty acids, in particular, fatty acids in the plastidic galactolipids in most plant species are made up of about 70-80% of the trienoic fatty acids, hexadecatrienoic and α-linolenic acids (16:3 and 18:3) (Harwood 1980). In soybean phosphatidylglycerol (PG) is the only lipid synthesized by the prokaryotic type pathway, one of the two glycerolipid synthetic pathways in plants. The other leaf glycerolipids, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), digalactosyldiacylglycerol (DGDG), and sulphoquinovosyldiacylglycerol (SQDG) are synthesized through the eukaryotic lipid pathway. Soybean lacks hexatrienoic acid (16:3) and contains α-linolenic (18:3) as the only trienoic fatty acid (Browse, Somerville, 1991). Omega-3 fatty acid desaturases are microsomal enzymes that catalyze the insertion of a third double bond into α-linoleic acid (18:2∆9, 12) to produce α-linolenic acid (18:3∆9, 12, 15). They, like the microsomal ω-6 desaturases, are characterized by the presence of a diiron cofactor that interacts with three conserved histidine motifs (Byfield & Upchurch, 2007B). Three soybean microsomal ω-6 desaturase genes have been isolated: *GmFAD3A*, *GmFAD3B*, and *GmFAD3C* (Bilyeu et al., 2003, Anai et al., 2005). *GmFAD3A* was found to be highly

In both soybean seed and leaf tissues, the levels of 18:2 and 18:3 gradually increase as temperature decreases to 18/12oC, but the levels of *GmFAD2-1*, *GmFAD2-2*, and *GmFAD6* transcripts were found not to increase at low temperature. This suggests that the elevated 18:2 and 18:3 in developing seeds grown at low temperature are not due to enhanced expression (transcriptional control) of these ω-6 genes (Heppard et al., 1996). On the other hand, in developing soybean seed, the levels of 18:2 and 18:3 decreases as temperature increases to 30/26oC and higher, and the levels of *GmFAD2-1A* and *2-1B* transcripts were found to decrease. This suggests transcriptional down-regulation of the *GmFAD2-1* genes does occur as growth temperatures increase (Byfield & Upchurch, 2007A). Substantial evidence suggests that post-translational regulatory mechanisms likely play an important role in modulating FAD2-1 enzyme activities. The FAD2-1A isoform was found to be more unstable than FAD2-1B, especially at elevated growth temperatures. In addition, the FAD2- 1s were phosphorylated during seed development. Evidence suggests that phosphorylation may down regulate FAD2-1 enzyme activity. Thus, growth at elevated temperature results in increased 18:1 and decreased 18:2 and 18:3 because the FAD2-1 oleate desaturase

Evidence for the participation of microsomal ω-6 fatty acid desaturases in the responses of plants to pathogen infection is not plentiful. Treatment of cultured parsley cells with the Pep25 peptide elicitor derived from the soybean oomycete pathogen *Phytophthora sojae* resulted in a strong local resistance response. Omega-6 fatty acid desaturase transcripts accumulated rapidly and transiently in elicitor-treated cells, protoplasts, and leaves, suggesting that 18:1 desaturation is an early component of the response of parsley to pathogen infection (Kirsch et al. 1997). Growth chamber experiments (Thomas et al., 2003, Xue et al., 2008) have shown that elevated growth temperatures (34/26 versus 22/18oC) during seed development results in higher 18:1 and reduced 18:2 content in seed lipid. Mature soybean seeds with higher ratios of 18:1 to 18:2 that were inoculated with the fungal pathogen *Cercospora kikuchii* were colonized more heavily by the fungus than inoculated

The membrane lipids of higher plants including soybean are characterized by a high proportion of polyunsaturated fatty acids, in particular, fatty acids in the plastidic galactolipids in most plant species are made up of about 70-80% of the trienoic fatty acids, hexadecatrienoic and α-linolenic acids (16:3 and 18:3) (Harwood 1980). In soybean phosphatidylglycerol (PG) is the only lipid synthesized by the prokaryotic type pathway, one of the two glycerolipid synthetic pathways in plants. The other leaf glycerolipids, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), digalactosyldiacylglycerol (DGDG), and sulphoquinovosyldiacylglycerol (SQDG) are synthesized through the eukaryotic lipid pathway. Soybean lacks hexatrienoic acid (16:3) and contains α-linolenic (18:3) as the only trienoic fatty acid (Browse, Somerville, 1991). Omega-3 fatty acid desaturases are microsomal enzymes that catalyze the insertion of a third double bond into α-linoleic acid (18:2∆9, 12) to produce α-linolenic acid (18:3∆9, 12, 15). They, like the microsomal ω-6 desaturases, are characterized by the presence of a diiron cofactor that interacts with three conserved histidine motifs (Byfield & Upchurch, 2007B). Three soybean microsomal ω-6 desaturase genes have been isolated: *GmFAD3A*, *GmFAD3B*, and *GmFAD3C* (Bilyeu et al., 2003, Anai et al., 2005). *GmFAD3A* was found to be highly

enzymes are substantially inactivated (Tang et al. 2005).

seeds with lower 18:1 to 18:2 ratios (Xue et. al., 2008).

**4. Soybean omega-3 linoleate fatty acid desaturases** 

expressed in seeds and *FAD3B* and *FAD3C* in both vegetative tissues and seeds. *GmFAD3A*, *B*, and *C* encode proteins that lack N-terminal chloroplast signal peptides. Soybean lines have been identified that produce low (2.8% compared to 8% for wild type) levels of 18:3 in their seed lipid. Low 18:3 in soybean seed lipid is a desired trait since 18:3 contributes to oil instability and rancidity. Molecular characterization of the low 18:3 line showed that a missplice mutation was present in *FAD3A* and also a single SNP altering a codon glycine to glutamic acid was present in *FAD3C (*Bilyeu et al., 2005). Molecular identity probes (CAPS markers, SNPs) were developed for all three soybean FAD3 genes and deployment of these probes for screening combinations of *FAD3* mutant alleles have allowed the development of new soybean lines with 1% 18:3 (Bilyeu et al., 2006, Beuselinck et al. 2006). Chloroplast localized soybean ω-6 fatty acid desaturase genes, designated *GmFAD7* and *GmFAD8* (after *Arabidopsis* chloroplast ω-6 desaturase functional nomenclature) have been partially characterized (Collados et al., 2006) and they do possess N-terminal chloroplast signal peptides (Table 3).


Table 3. Soybean (*Glycine max* L.) omega-3 fatty acid desaturase genes including putative chromosome and linkage group assignment, and tissue transcript expression.

The discussion that follows focuses mainly on regulation of ω-3 FAD activity at the level of transcription control. A recent report has provided compelling evidence for a temperaturesensitive post-translational regulation of FAD3 protein abundance that involves a combination of cis-acting degradation signals and the ubiquitin-protease pathway that modulates FAD3 protein amounts in response to temperature (O'Quin et al., 2010). The halflife of FAD3 protein is greater at cooler temperatures and protein degradation required specific components of the endoplasmic reticulum protease pathway.

Most of our understanding of ω-3 FAD activity and stress acclamation in plants, including temperature change and pathogen infection, comes from research with *Arabidopsis* and other plants. Characterization of *AtFAD7* gene sequence revealed an open reading frame of 1338 bp comprised of 8 exons that encoded a deduced 446 amino acid peptide of 51.1 kDa. Growth temperature had no apparent effect on the steady-state levels of *FAD7* transcripts in wild-type plants (Nishiuchi & Iba, 1998). The *AtFAD8* sequence was found to code for a 435 amino acid peptide of 50.1 kDa that also contained a consensus chloroplast transit peptide. The coding region of *AtFAD8* shared 75% nucleotide identity with *AtFAD7*. Transcript

Soybean Fatty Acid Desaturation Pathway:

Responses to Temperature Changes and Pathogen Infection 121

upregulated expression of the birch ω-3 desaturases, *BpFAD3*, *BpFAD7* and *BpFAD8* (Martz et al. 2006). Transgenic tomato plants in which the microsomal omega-3 desaturases have been silenced have greatly reduced *LeFAD3* transcripts, contain low levels of 18:3, higher levels of 18:2, and exhibit long-term heat tolerance at 36oC (Wang et al. 2010). Heat stressed *LeFAD3*-suppressed plants produced greater fresh weight of aerial plant parts and had a more intact chloroplast membrane structure than did heat stressed wild-type plants. Growth of soybean plants at a cool temperature (22/18oC, D/N) during seed development resulted in elevated seed 18:3 and elevated Gm*FAD3A*, *FAD3B* and *FAD3C* transcript expression, and conversely, decreased 18:3 and transcript expression of these microsomal omega-3 desaturase genes at a warm temperature (30/26oC, D/N) during seed development (Byfield and Upchurch 2007B). A general conclusion to be drawn from experiments with *Arabidopsis* and other plants is that transcript expression of *FAD8* and *FAD3* change in response to changes in ambient temperature, and *FAD8* is cold-inducible whereas expression of *FAD7* is not affected by changes in temperature (McConn et al., 1994, Berberich et al., 1998, Iba, 2002, Upchurch & Byfield, 2007, Nair et al., 2009, Wang et al., 2010). Another conclusion is that the increased 18:3 level in chloroplast membranes due to upregulated *FAD8* expression is associated with low temperature tolerance in *Arabidopsis* and other plants. Presumably,

temperature regulation of soybean *GmFAD 7* and *FAD8* follows a similar pattern.

Upregulation of *FAD7* and increased 18:3 levels in chloroplasts have physiological roles in modulating plant defense responses to pathogens in several plant-pathogen systems. For instance, *FAD7* has been shown to be required to provide 18:3 for the synthesis of a longdistance signal (not jasmonic acid) that is required for the induction of systemic acquired resistance (SAR) in *Arabidopsis* and tomato (Chaturvedi et al. 2008). The *A. thaliana FAD7 and FAD8* double mutation prevents the synthesis of trienoic acids in chloroplast lipids, causing a reduction in the production and accumulation of reactive oxygen intermediates in leaves, reduced levels of programmed cell death, and compromised resistance to several avirulent *Pseudomonas syringae* strains (Yaeno et al. 2004). On the other hand and in contrast, disease resistance to compatible and incompatible races of the rice blast fungus *Magnaporthe grisea* is enhanced in 18:2 accumulating and 18:3-deficient transgenic rice (F78Ri) in which *OsFAD7* and *OsFAD8* were suppressed. The 18:3 Jasmonate-mediated wound responses were suppressed, but the expression of jasmonate-responsive PR genes, PBZ1 and PR1b were induced after inoculation. In rice F78Ri mutant plants, the 18:2-derived hydroperoxides and hydroxides (HPODEs and HODEs) increased significantly and these molecules inhibited the growth of *M. grisea* more strongly than their 18:3-derived counterparts (Yara et al., 2007, 2008). In *Arabidopsis*, local mechanical wounding and pathogen attack causes a rapid rise of *AtFAD7* transcripts in the basal rosette leaves and induces *AtFAD7* expression in the roots. Inhibitors of the oxylipin octadecanoid pathway strongly suppress wound activation of the *FAD7* promoter in roots but not in leaves and stems (Nishiuchi et al., 1997). A specific region of the *AtFAD7* promoter is required for wound-activated expression of this gene in leaves and stems, while another region is necessary for wound-activated, jasmonic acid-responsive expression of the gene in roots (Nishiuchi et al., 1999) suggesting that a jasmonateindependent wound signal may induce the activation of the *FAD7* gene in leaves and stems. In tomato *(Lycopersicon esculentum*) containing a mutation in *Spr2* (which encodes the chloroplast ω-3 FAD gene, *LeFAD7*), the 18:3 content of the leaves was less than 10% of wildtype levels. The accumulation of hexadecatrienoic acid was also abolished and both woundinduced jasmonic acid biosynthesis and the production of a long-distance signal for expression of defensive genes were reduced such that *Spr2* plants were compromised in

abundance of *AtFAD8* strongly increases in plants grown at low temperatures suggesting that the role of *FAD8* in *Arabidopsis* is to provide increased chloroplast membrane 18:3+16:3 in plants that are exposed to low growth temperature (Nishiuchi & Iba, 1998). The temperature dependent regulation of *AtFAD8* expression is not due to the *FAD8* 5' flanking region (promoter and untranslated region), but to the exon/intron structure that is inherent in the *AtFAD8* gene (Iba, 2002). Examination of *GmFAD7* and *GmFAD8* at NCBI GenBank accession numbers HM769340 and HM769341 revealed that both soybean genes have a similar structure containing 8 exons ranging in size from 67 to 521 nucleotides and 7 introns ranging in size from 90 to 393 nucleotides. The soybean *FAD7* and *FAD8* intron/exon structure is similar to *FAD7* and *FAD8* structures of other higher plants except that the rice *OsFAD8* contains 7 exons. For both soybean genes, an exonic sequence of 1362 base pairs encodes a predicted protein of 453 amino acid residues with molecular masses of 51.3 and 51.4 kDa, respectively, for *GmFAD7* and *GmFAD8*. Using *GmFAD7* and *GmFAD8* genomic sequences as queries to interrogate the Williams 82 genome database (Schmutz et al., 2010) revealed that each gene was present in the Williams 82 genome as two complete copies located on different chromosomes, one *GmFAD7* copy located on chromosome 18 and a second on chromosome 7. A recent report has shown that the second *GmFAD7* gene, designated *GmFAD7-2*, and located on chromosome 7 is paralogous to *GmFAD7-1* located on chromosome 18 (Andreu et al., 2010). The paralogous nature of *GmFAD7-1* and *GmFAD7- 2* is supported by the finding of specific gene-related FAD7 protein conformations in soybean seeds. The FAD7 protein conformations were differentially affected by *in vitro* changes in redox conditions and iron availability suggesting the existence of tissue-specific post-translational mechanisms that affect the distribution and activity of the FAD7 enzymes. Two complete copies of *GmFAD8* were also present in the Williams 82 genome sequence and they may, as well, be paralogous. One is located on chromosome 3 and the second on chromosome 1. Recent studies have characterized soybean FAD7 chloroplast localization and the transcript expression patterns in response to light of both microsomal and plastidal ω-3 soybean desaturases. In situ analysis using confocal microscopy with FAD7 antibody and chlorophyll auto fluorescence has shown that the soybean FAD7 protein is preferentially localized to the chloroplast thylakoid membranes suggesting that not only the chloroplast envelope, but also the thylakoid membranes could be sites of lipid desaturation in higher plants (Andreu et al., 2007). *GmFAD3, GmFAD7*, and *GmFAD8* transcription and transcript stability have been found to be differentially regulated by light (Collados et al., 2006). In soybean cell suspension, darkness leads to an overall decrease in 18:3 levels and *GmFAD3* and *GmFAD8* transcripts are undetectable, but after reillumination *FAD3* and *FAD8* transcript abundance increased concomitant with an increase in 18:3 accumulation. *GmFAD7* transcript levels were remarkably similar under dark or light conditions and *GmFAD7* mRNA stability dramatically increased in the dark as well. FAD7 protein levels were also very stable in either light or dark conditions, suggesting that an additional posttranslational regulatory mechanism may control the activity of FAD7 in response to light. Numerous studies have shown that temperature regulates the transcript expression of

plastid ω-3 and microsomal ω-3 desaturases and leaf trienoic fatty acid levels in plants. In *Brassica napus* leaf 16:3/18:3 levels increase in MGDG during low temperature acclimation (Williams et al. 1996). In birch (*Betula pendula*) seedlings exposed to low temperatures (+ 4 to -24oC) increased 18:3 in the chloroplast membrane lipids (MGDG, DGDG and PG) were found in the leaves at colder temperatures. The higher 18:3 levels were associated with

abundance of *AtFAD8* strongly increases in plants grown at low temperatures suggesting that the role of *FAD8* in *Arabidopsis* is to provide increased chloroplast membrane 18:3+16:3 in plants that are exposed to low growth temperature (Nishiuchi & Iba, 1998). The temperature dependent regulation of *AtFAD8* expression is not due to the *FAD8* 5' flanking region (promoter and untranslated region), but to the exon/intron structure that is inherent in the *AtFAD8* gene (Iba, 2002). Examination of *GmFAD7* and *GmFAD8* at NCBI GenBank accession numbers HM769340 and HM769341 revealed that both soybean genes have a similar structure containing 8 exons ranging in size from 67 to 521 nucleotides and 7 introns ranging in size from 90 to 393 nucleotides. The soybean *FAD7* and *FAD8* intron/exon structure is similar to *FAD7* and *FAD8* structures of other higher plants except that the rice *OsFAD8* contains 7 exons. For both soybean genes, an exonic sequence of 1362 base pairs encodes a predicted protein of 453 amino acid residues with molecular masses of 51.3 and 51.4 kDa, respectively, for *GmFAD7* and *GmFAD8*. Using *GmFAD7* and *GmFAD8* genomic sequences as queries to interrogate the Williams 82 genome database (Schmutz et al., 2010) revealed that each gene was present in the Williams 82 genome as two complete copies located on different chromosomes, one *GmFAD7* copy located on chromosome 18 and a second on chromosome 7. A recent report has shown that the second *GmFAD7* gene, designated *GmFAD7-2*, and located on chromosome 7 is paralogous to *GmFAD7-1* located on chromosome 18 (Andreu et al., 2010). The paralogous nature of *GmFAD7-1* and *GmFAD7- 2* is supported by the finding of specific gene-related FAD7 protein conformations in soybean seeds. The FAD7 protein conformations were differentially affected by *in vitro* changes in redox conditions and iron availability suggesting the existence of tissue-specific post-translational mechanisms that affect the distribution and activity of the FAD7 enzymes. Two complete copies of *GmFAD8* were also present in the Williams 82 genome sequence and they may, as well, be paralogous. One is located on chromosome 3 and the second on chromosome 1. Recent studies have characterized soybean FAD7 chloroplast localization and the transcript expression patterns in response to light of both microsomal and plastidal ω-3 soybean desaturases. In situ analysis using confocal microscopy with FAD7 antibody and chlorophyll auto fluorescence has shown that the soybean FAD7 protein is preferentially localized to the chloroplast thylakoid membranes suggesting that not only the chloroplast envelope, but also the thylakoid membranes could be sites of lipid desaturation in higher plants (Andreu et al., 2007). *GmFAD3, GmFAD7*, and *GmFAD8* transcription and transcript stability have been found to be differentially regulated by light (Collados et al., 2006). In soybean cell suspension, darkness leads to an overall decrease in 18:3 levels and *GmFAD3* and *GmFAD8* transcripts are undetectable, but after reillumination *FAD3* and *FAD8* transcript abundance increased concomitant with an increase in 18:3 accumulation. *GmFAD7* transcript levels were remarkably similar under dark or light conditions and *GmFAD7* mRNA stability dramatically increased in the dark as well. FAD7 protein levels were also very stable in either light or dark conditions, suggesting that an additional posttranslational regulatory mechanism may control the activity of FAD7 in response to light. Numerous studies have shown that temperature regulates the transcript expression of plastid ω-3 and microsomal ω-3 desaturases and leaf trienoic fatty acid levels in plants. In *Brassica napus* leaf 16:3/18:3 levels increase in MGDG during low temperature acclimation (Williams et al. 1996). In birch (*Betula pendula*) seedlings exposed to low temperatures (+ 4 to -24oC) increased 18:3 in the chloroplast membrane lipids (MGDG, DGDG and PG) were found in the leaves at colder temperatures. The higher 18:3 levels were associated with upregulated expression of the birch ω-3 desaturases, *BpFAD3*, *BpFAD7* and *BpFAD8* (Martz et al. 2006). Transgenic tomato plants in which the microsomal omega-3 desaturases have been silenced have greatly reduced *LeFAD3* transcripts, contain low levels of 18:3, higher levels of 18:2, and exhibit long-term heat tolerance at 36oC (Wang et al. 2010). Heat stressed *LeFAD3*-suppressed plants produced greater fresh weight of aerial plant parts and had a more intact chloroplast membrane structure than did heat stressed wild-type plants. Growth of soybean plants at a cool temperature (22/18oC, D/N) during seed development resulted in elevated seed 18:3 and elevated Gm*FAD3A*, *FAD3B* and *FAD3C* transcript expression, and conversely, decreased 18:3 and transcript expression of these microsomal omega-3 desaturase genes at a warm temperature (30/26oC, D/N) during seed development (Byfield and Upchurch 2007B). A general conclusion to be drawn from experiments with *Arabidopsis* and other plants is that transcript expression of *FAD8* and *FAD3* change in response to changes in ambient temperature, and *FAD8* is cold-inducible whereas expression of *FAD7* is not affected by changes in temperature (McConn et al., 1994, Berberich et al., 1998, Iba, 2002, Upchurch & Byfield, 2007, Nair et al., 2009, Wang et al., 2010). Another conclusion is that the increased 18:3 level in chloroplast membranes due to upregulated *FAD8* expression is associated with low temperature tolerance in *Arabidopsis* and other plants. Presumably, temperature regulation of soybean *GmFAD 7* and *FAD8* follows a similar pattern. Upregulation of *FAD7* and increased 18:3 levels in chloroplasts have physiological roles in modulating plant defense responses to pathogens in several plant-pathogen systems. For instance, *FAD7* has been shown to be required to provide 18:3 for the synthesis of a longdistance signal (not jasmonic acid) that is required for the induction of systemic acquired resistance (SAR) in *Arabidopsis* and tomato (Chaturvedi et al. 2008). The *A. thaliana FAD7 and FAD8* double mutation prevents the synthesis of trienoic acids in chloroplast lipids, causing a reduction in the production and accumulation of reactive oxygen intermediates in leaves, reduced levels of programmed cell death, and compromised resistance to several avirulent *Pseudomonas syringae* strains (Yaeno et al. 2004). On the other hand and in contrast, disease resistance to compatible and incompatible races of the rice blast fungus *Magnaporthe grisea* is enhanced in 18:2 accumulating and 18:3-deficient transgenic rice (F78Ri) in which *OsFAD7* and *OsFAD8* were suppressed. The 18:3 Jasmonate-mediated wound responses were suppressed, but the expression of jasmonate-responsive PR genes, PBZ1 and PR1b were induced after inoculation. In rice F78Ri mutant plants, the 18:2-derived hydroperoxides and hydroxides (HPODEs and HODEs) increased significantly and these molecules inhibited the growth of *M. grisea* more strongly than their 18:3-derived counterparts (Yara et al., 2007, 2008). In *Arabidopsis*, local mechanical wounding and pathogen attack causes a rapid rise of *AtFAD7* transcripts in the basal rosette leaves and induces *AtFAD7* expression in the roots. Inhibitors of the oxylipin octadecanoid pathway strongly suppress wound activation of the *FAD7* promoter in roots but not in leaves and stems (Nishiuchi et al., 1997). A specific region of the *AtFAD7* promoter is required for wound-activated expression of this gene in leaves and stems, while another region is necessary for wound-activated, jasmonic acid-responsive expression of the gene in roots (Nishiuchi et al., 1999) suggesting that a jasmonateindependent wound signal may induce the activation of the *FAD7* gene in leaves and stems. In tomato *(Lycopersicon esculentum*) containing a mutation in *Spr2* (which encodes the chloroplast ω-3 FAD gene, *LeFAD7*), the 18:3 content of the leaves was less than 10% of wildtype levels. The accumulation of hexadecatrienoic acid was also abolished and both woundinduced jasmonic acid biosynthesis and the production of a long-distance signal for expression of defensive genes were reduced such that *Spr2* plants were compromised in

Soybean Fatty Acid Desaturation Pathway:

impact plant defenses.

the manuscript.

**7. References**

1623.

447-452.

1336-1344.

Vol. 43: 1833-1838.

**6. Acknowledgements** 

Responses to Temperature Changes and Pathogen Infection 123

acids in plant lipid, global climate change (Garrett et al. 2006) may potentially negatively

I thank Dr. Ralph E. Dewey, Crop Science Department, North Carolina State University, Raleigh for helpful discussions on the soybean omega-6 fatty acid desaturase alleles and Dr. Martha E. Ramirez, ARS Soybean & Nitrogen Fixation Unit, Raleigh for a critical reading of

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defense against attack by tobacco hornworm (Li et al. 2003). Recently it was reported that silencing of the three soybean GmFAD3 genes enhanced the accumulation of *Bean Pod mottle virus* (BPMV) in plant tissues and enhanced susceptibility to virulent *Pseudomonas syringae*  bacteria (Singh et al. 2011). Silenced plants exhibited increased levels of jasmonic acid and slightly reduced levels of 18:3 indicating that loss of microsomal ω-3 activity enhances jasmonate accumulation and thereby susceptibility to *BPMV* in soybean.
