**2. Soybean delta-9 stearoyl-acyl carrier protein-desaturases**

The ∆-9 stearoyl-acyl carrier protein-desaturases are soluble enzymes localized to the stroma fraction of plastids that introduce the first double bond into stearoyl-ACP (18:0-ACP) to produce oleoyl (18:1∆9)-ACP. Delta 9-stearoyl-ACP-desaturases thus occupy a key position in C18 fatty acid biosynthesis since perturbation of SACPD gene expression and/or enzyme activity may modulate the relative cellular content of both stearate and oleate. Three alleles of *SACPD* have been identified and characterized from soybean (Table 1).


Table 1. Soybean (*Glycine max* L.) ∆9-stearoyl-acyl carrier protein-desaturase genes including putative chromosome and linkage group assignment and tissue transcript expression.

mechanism, the modification of membrane fluidity in response to temperature stress results in the maintenance of a membrane environment suitable for the function of critical integral proteins, such as the photosynthetic machinery in chloroplasts (Nishiuchi et al., 1998). The fatty acid composition in soybean tissues is, in addition, responsive to biotic (pathogen) attack (Iba, 2002, Upchurch, 2008) and fatty acids and fatty acid-derived compounds act as signals of plant defense gene expression (Kachroo et al., 2001, Weber, 2002). Evidence suggests that the levels of 18:0 and 18:1 are critical for defense against pathogens in soybean as they have been shown to be in *Arabidopsis thaliana* (Kachroo & Kachroo, 2009). Moreover, the oleate and linoleate content of soybean seeds appears to influence the course of seed

Plants often encounter temperatures that are stressing, as well as pathogens and insects in the environment, sometimes simultaneously. Thus, the current worldwide situation of diminishing farm land and the heightened effects of global climate change on the productivity of agriculture (Garrett et al., 2006) increase the need to understand stress responses in crop plants such as soybean. More complete knowledge of fatty acid metabolism and its regulation in this and other important oilseed crops may significantly aid the development of effective strategies for managing abiotic and biotic stresses in the agricultural environment. This chapter focuses on a concise description of three fatty acid desaturase gene families and their contributions to the acclimation of soybean and other plants to high and low temperature and pathogen infection. Investigations of the regulation of desaturase expression and activity by temperature and pathogens are relatively recent in soybean and current results suggest complexity, yet a basic understanding of these phenomena are required if varieties are to be developed that possess stable and durable

The ∆-9 stearoyl-acyl carrier protein-desaturases are soluble enzymes localized to the stroma fraction of plastids that introduce the first double bond into stearoyl-ACP (18:0-ACP) to produce oleoyl (18:1∆9)-ACP. Delta 9-stearoyl-ACP-desaturases thus occupy a key position in C18 fatty acid biosynthesis since perturbation of SACPD gene expression and/or enzyme activity may modulate the relative cellular content of both stearate and oleate. Three alleles

> Chromosome, Linkage Group

Transcript

and seeds

and seeds

seeds

expression References

Byfield et al., 2006 Zhang et al., 2008 Ha et al., 2010

colonization by a fungal pathogen (Xue et al., 2008).

expression of desirable stress-acclimation traits.

function Gene name GenBank

Enzyme

∆9-Stearoyl-ACP-Desaturase

**2. Soybean delta-9 stearoyl-acyl carrier protein-desaturases** 

of *SACPD* have been identified and characterized from soybean (Table 1).

accession

*GmSACPD-A* AY885234 7, M Vegetative

*GmSACPD-B* AY885233 2, D1b Vegetative

*GmSACPD-C* EF113911 14, B2 Highly in

Table 1. Soybean (*Glycine max* L.) ∆9-stearoyl-acyl carrier protein-desaturase genes including putative chromosome and linkage group assignment and tissue transcript expression.

Transcripts of the *GmSACPD-A* and *-B* were detected in developing seeds and other tissues, but differences in transcript abundance between *-A* and *-B* were not dramatic (Byfield et al., 2006). Translation of the 1158-bp transcript of *SACPD-A* or *-B* predicts a protein of 411 amino acids with a molecular mass of 47.2 kDa. The enzyme is a homodimer with each mature subunit containing an independent binuclear iron cluster. Soybean *SACPD*s contain two characteristic histidine box motifs. High transcript levels of a unique third allele, *GmSACPD-C*, is expressed only in developing seeds (Zhang et al., 2008). Structurally, *SACPD-C* is composed of two exons, not three as for *SACPD-A* and *-B*, separated by an 846 bp intron. Thus, *SACPD-C* differs from the *SACPD-A* and *-B* alleles in that it lacks their large intron located immediately after the putative transit peptide-encoding region. Mutations at *SACPD-C* in two soybean germplasm sources, the mutants A6 (30% 18:0) and FAM94-41 (9% 18:0) (Pantalone et al., 2002), have decreased *SACPD-C* expression and elevated seed stearic acid levels. This finding suggests, conversely, that germplasm with high SACPD-C gene expression and/or enzyme activity would produce elevated 18:1 levels. Polymerase chain reaction-based CAPS (Cleaved Amplified Polymorphism) gene probes (Zhang et al., 2008) were developed to screen soybean germplasm for mutations at *SACPD-C*, since varieties with elevated stearate are desirable for certain industrial uses such as food shortening and soap making.

The effect of increasing temperatures (from 22/18oC to 30/26oC) during seed development on 18:0 accumulation and *SACPD-A* and -*B* transcript accumulation has been measured in growth chamber environments (Byfield and Upchurch, 2007A). At the cool temperature, transcript accumulation of both *SACPD-A* and *-B* was significantly elevated and significantly decreased at the warmer temperature. Decreased *SACPD-A* and *-B* transcript accumulation at the warmer temperature was positively associated with a significant increase in the level of seed 18:0, but only in the high stearate mutant A6. It was suggested that temperature modulation of 18:0 content in wild type soybeans may be more complex, potentially involving in addition to the *SACPDs*, plastid thioesterase *FAT* genes, or warmtemperature post-translational modulation of SACPD enzyme activity.

The role of fatty acid desaturation pathways in mediating pathogen defense signaling has been, until recently, examined mainly in *Arabidopsis*. The *SSI2* gene cloned from *Arabidopsis* was shown to encode an (*At*) ∆9-stearoyl-ACP-desaturase. Plants with the recessive mutation *ssi2* had a 10-fold reduction in SACPD enzyme content resulting in elevated 18:0 and reduced 18:1 content. Reduced SACPD activity in the *ssi2* mutant lead to induction of a salicylic acid-signaled defense response to the oomycete *Peronospora parasitica,* plant dwarfing and spontaneous leaf lesion formation, but also to inhibition of the jasmonic acidsignaled defense response to the fungus *Botrytis cinerea* (Kachroo et al., 2001, Nandi et al., 2003, Kachroo et al., 2005, Kachroo et al., 2007). In a situation similar to that of *Arabidopsis*, suppression of the rice fatty-acid desaturase gene *OsSSI2* (a rice ∆9-stearoyl-ACPdesaturase) by transposon insertion or RNAi-mediated knockdown increased 18:0 and reduced 18:1 in plants and markedly enhanced resistance to the blast fungus *Magnaporthe grisea* and the leaf blight bacterium *Xanthomonas oryzae* pv. *oryzae* (Jiang et al., 2009). On the other hand, multiple stresses imposed on avocado fruits including inoculation with the fungal pathogen *Colletotrichum gloeosporioides*, exposure to ethylene, C02, fruit wounding, and low temperature exposure increased transcript abundance of avocado (*Av*) ∆9-stearoyl-ACP-desaturase. The up-regulation of *AvSACPD* was accompanied by increases in the concentration of 18:2 (presumably from increased 18:1), increase in an antifungal diene volatile and enhanced resistance to fungal infection (Madi et al., 2003). In soybean as in

Soybean Fatty Acid Desaturation Pathway:

Missouri in the US and one in Costa Rica.

Responses to Temperature Changes and Pathogen Infection 117

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

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