**3. Soybean omega-6 oleate fatty acid desaturases**

The soybean ω-6 oleate fatty acid desaturases (FAD2s) are microsomal enzymes that initiate the primary route of polyunsaturated lipid biosynthesis by catalyzing the first extraplastidal desaturation to convert 18:1 esterified to phosphatidylcholine to α-18:2 (Heppard et al., 1996). Omega-6 desaturase enzymes are typical of other microsomal desaturases in that they contain three histidine box motifs, possess a C-terminal signal for endoplasmic reticulum retention (Li et al. 2007) and have four predicted transmembrane spanning domains (Tang et al., 2005). Four different soybean ω-6 desaturase genes comprise the soybean *FAD2* gene family (Schlueter et. al., 2007) including *GmFAD2-1* and *GmFAD2-2* and their alleles (Heppard et al., 1996, Tang et al., 2005, Bachlava et. al., 2009), *GmFAD2-3* (Li et al. 2007), and *GmFAD6* (Heppard et al., 1996, Bachlava et al., 2009) (Table 2).


Table 2. Soybean (*Glycine max* L.) omega-6 fatty acid desaturase genes including putative chromosome assignment and tissue transcript expression.

GmFAD2-1 genes have a short intron immediately after the start ATG which is spliced out and their mature transcripts encode proteins of approximately 387 amino acids (Tang et. al., 2005). *GmFAD2-1s* are highly expressed during lipid synthesis in developing seeds and not in vegetative tissues, while *GmFAD2-2*s are constitutively expressed in both vegetative tissue and developing seeds. Although the FAD2-2s contribute to the production of 18:1 in all tissues, transcript expression analysis suggests that the FAD2-1s play the major role in

*Arabidopsis*, silencing of the *SACPD* genes (*-A*,-*B*, and -*C*) by a *Bean pod mottle virus*-based vector resulted in plants with reduced 18:1, elevated 18:0, the formation of spontaneous lesions, increased salicylic acid accumulation, and constitutively expressed pathogenesisrelated genes. These plants also exhibited enhanced resistance to bacterial and oomycete

The soybean ω-6 oleate fatty acid desaturases (FAD2s) are microsomal enzymes that initiate the primary route of polyunsaturated lipid biosynthesis by catalyzing the first extraplastidal desaturation to convert 18:1 esterified to phosphatidylcholine to α-18:2 (Heppard et al., 1996). Omega-6 desaturase enzymes are typical of other microsomal desaturases in that they contain three histidine box motifs, possess a C-terminal signal for endoplasmic reticulum retention (Li et al. 2007) and have four predicted transmembrane spanning domains (Tang et al., 2005). Four different soybean ω-6 desaturase genes comprise the soybean *FAD2* gene family (Schlueter et. al., 2007) including *GmFAD2-1* and *GmFAD2-2* and their alleles (Heppard et al., 1996, Tang et al., 2005, Bachlava et. al., 2009), *GmFAD2-3* (Li et

> Chromosom e, linkage group

*1A* AB188250 20, I Highly in

*1B* AB188251 10, O Highly in

*2A* AB188252 19, L Vegetative

*2B* AB188253 19, L Vegetative

*2C* AC166742.25 15, E Vegetative

*2D* AC166091.3 3, N Vegetative

*GmFAD2-3* DQ53237 3 ,N Vegetative

*GmFAD6* L29215 2, D1b chloroplasts Table 2. Soybean (*Glycine max* L.) omega-6 fatty acid desaturase genes including putative

GmFAD2-1 genes have a short intron immediately after the start ATG which is spliced out and their mature transcripts encode proteins of approximately 387 amino acids (Tang et. al., 2005). *GmFAD2-1s* are highly expressed during lipid synthesis in developing seeds and not in vegetative tissues, while *GmFAD2-2*s are constitutively expressed in both vegetative tissue and developing seeds. Although the FAD2-2s contribute to the production of 18:1 in all tissues, transcript expression analysis suggests that the FAD2-1s play the major role in

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seeds

seeds

and seeds

and seeds

and seeds

and seeds

and seeds

expression References

Heppard et al., 1996, Tang et al. 2005, Bachlava et al. 2009, Li et al., 2007, Ha et al., 2010

al. 2007), and *GmFAD6* (Heppard et al., 1996, Bachlava et al., 2009) (Table 2).

accession

pathogens (Kachroo et al., 2008, Kachroo & Kachroo, 2009).

**3. Soybean omega-6 oleate fatty acid desaturases** 

Enzyme

Omega (ω)-6 Fatty Acid Desaturas e

function Gene name GenBank

*GmFAD2-*

*GmFAD2-*

*GmFAD2-*

*GmFAD2-*

*GmFAD2-*

*GmFAD2-*

chromosome assignment and tissue transcript expression.

the conversion of 18:1 to 18:2 in developing seeds. Two seed specific isoforms of FAD2-1, FAD2-1A and FAD2-1B, have been described that differ in stability at elevated temperature (Tang et al., 2005). Recent soybean genomic analysis has shown that *FAD2-2* exists as four alleles, *GmFAD2-2A, 2-2B*, *2-2C*, and *2-2D* (Schlueter et al., 2007, Bachlava et al., 2009, Ha et al., 2010). The expression level of *GmFAD2*-2C has been shown to increase eightfold in developing pods grown at 18/12oC in comparison to those grown at 32/28oC. The third gene, *GmFAD2-3*, is also constitutively expressed in both vegetative and developing seed tissues but shows no significant changes in transcript abundance in cold stressed leaves (Li et al., 2007). The fourth gene, *GmFAD6,* encodes an omega-6 desaturase that localizes to the plastid membrane. The expression pattern of the FAD6 gene does not suggest changes in transcript abundance in response to different temperatures (Heppard et al., 1996).

Significant efforts have been expended to select soybean varieties that produce higher seed oil 18:1 content, for example, mid-oleic soybean line N98-4445A which produces 50-60% 18:1 as a percent of total seed lipid fatty acids (Burton et al., 2005). Our understanding of the phenomena of elevated seed oleate and efforts to develop soybeans with this phenotype have been facilitated by the isolation and characterization of the X-ray induced mutant M23 and others with similar oleate phenotypes (Takagi, Rahman, 1996, Anai et al. 2008) and the earlier molecular characterizations of *FAD2-1* in high-oleate producing peanut mutants (Martinez-Rivas et al., 2001, Lopez et al., 2002). M23 was found to contain a large genomic lesion that completely deleted *GmFAD2-1A* (Alt et al., 2005, Sandhu et al., 2007) and mutant KK21 has a deletion of 232-bp downstream of the *FAD2-1A* ATG initiation codon (Anai et al., 2008). Both mutants produce 50-60% 18:1 in their seed lipid compared to approximately 20% 18:1 for conventional soybean cultivars. Many of the higher oleate soybean lines under development are progeny of crosses with the M23 mutant. Field trials have uncovered environmental instability in the expression of this trait in the M23-derived lines (Oliva et al., 2006, Scherder et al., 2008), as well as reductions in seed yield, protein, and oil (Scherder & Fehr, 2008). Possibly, the large genomic deletion in M23 (which extends outside of *FAD2-1A*) or additional X-ray induced mutations in M23 may be responsible for some or all of these additional phenotypic alterations. To develop soybean lines with more stable expression of elevated 18:1 without yield penalty, additional approaches involving reverse genetics have been applied. Ribozyme termination cassettes were employed with the aim of producing transgenic soybean with down-regulated GmFAD2-1 gene expression. Soybean transformants were recovered that stably displayed 18:1 levels in seed lipids of over 75% (Buhr et al., 2002). An intron sense suppression construct of *GmFAD2-1A* was employed with the aim of specifically reducing *FAD2-1* transcripts in developing seeds (Mroczka et al., 2010). Single copy transformants were recovered in which both *FAD2-1* alleles were suppressed that produced seeds with 18:1 levels elevated to 65 to 70% and corresponding reduction of 18:2. Targeting Induced Local Lesions In Genomes (TILLING) was employed with the aim of producing mutations in *GmFAD2-1A*. A missense amino acid mutation was recovered that resulted in an increase in seed 18:1 and a decrease in 18:2 compared to the wild type Williams 82 cultivar (Dierking & Bilyeu, 2009). Recently, soybean lines were identified that contain a single missense mutation in *GmFAD2-1A* or in *GmFAD2-1B* as a result of unique single nucleotide polymorphisms (SNPs) that were predicted to alter seed 18:1 content. Crosses were made to combine the two mutant FAD2-1 alleles from these otherwise conventional lines (Pham et al. 2010). Progeny homozygous for both mutant alleles consistently produced 80% seed 18:1 at different geographic locations, two in Missouri in the US and one in Costa Rica.

Soybean Fatty Acid Desaturation Pathway:

Gene name GenBank accession

*GmFAD7* HM769340 18, G and 7,

*GmFAD8* HM769341 3, N and 1,

specific components of the endoplasmic reticulum protease pathway.

chromosome and linkage group assignment, and tissue transcript expression.

peptides (Table 3).

Enzyme function

Omega (ω)-3 Fatty Acid Desaturase

Responses to Temperature Changes and Pathogen Infection 119

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

> Chromosome, linkage group

*GmFAD3A* AY204710 14, B2 Seeds highly,

*GmFAD3B* AY204711 2, D1b Vegetative

*GmFAD3C* AY204712 18, G Vegetative

Table 3. Soybean (*Glycine max* L.) omega-3 fatty acid desaturase genes including putative

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

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

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vegetative

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and seeds

<sup>M</sup>chloroplasts

D1a chloroplasts

expression References

Bilyeu et al., 2003, Collados et al., 2006, Ha et al., 2010, Upchurch & Ramirez, 2011

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

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