**1. Introduction**

112 Soybean – Genetics and Novel Techniques for Yield Enhancement

Yeh, C. & Sinclair, J. (1980). Sporulation and variation in size of conidia and conidiophores

ISSN 0191-2917

among five isolates of *Cercospora kikuchii*. *Plant Disease*, Vol.64, No.4, pp. 373-374,

Soybean [Glycine max (L.) Merr] is the largest oilseed crop produced and consumed worldwide, accounting for 58% of the world oilseed production (SoyStats, 2011), yet the oil produced from most available cultivars is still lacking in several quality characteristics. For example, the oil is too low in oleate and/or too high in linolenate content with resulting negative impacts on oil stability and human nutrition. Three fatty acid metabolism enzymes, the stearoyl-acyl carrier protein-desaturases (encoded by the *GmSACPD* genes), the omega-6 desaturases (*GmFAD2s*), and the omega-3 (*GmFAD3s*) desaturases largely determine the relative degree of unsaturated fatty acids and the content of the C18 fatty acids stearate (18:0), oleate (18:1), linoleate (18:2), and linolenate (18:3) in vegetative and seed lipids. In vitro studies have shown that it is possible to redesign soluble fatty acid desaturases from plants for altered fatty acid substrate and double bond position (Cahoon et al., 1997, Whittle et al., 2005) and in that way potentially alter the fatty acid content of plant lipids. Since the fatty acid composition of seed lipid is such an important determinate of oil quality, intensive efforts have also been mounted to select advantageous desaturase alleles (Wilson et al., 2001, Rajcan et al., 2005) and to manipulate molecularly desaturase expression and activity (Buhr et al., 2002), the goal being to produce elite soybean varieties with enhanced oil traits for the needs of industry and for improved human nutrition.

Both field and growth chamber experiments have shown that the fatty acid composition in soybean tissues is responsive to environmental temperature. In field studies, temperatures during the growing season affected seed linolenic content most clearly (Hou et al., 2006). Experiments to model climate change by increasing temperatures and [CO2] in controlled environment chambers (Thomas et al., 2003) showed that exposure to increasing [CO2] had no measurable effect, but higher temperatures (greater than 32/22oC day/night) reduced total seed oil concentration while oleate increased and linolenate decreased with increasing temperature. Transcripts of β-glucosidase, a gene expressed during seed development, was detected in seeds grown at 28/18oC but not detected in seeds grown at 40/30oC. This observation suggested that one mechanism by which climate change may affect soybean seed development is through the regulation of gene transcription. The ability to adjust membrane lipid fluidity by changing the levels of unsaturated fatty acids is provided mainly by the regulated activity of fatty acid desaturases (Iba 2002). Through this

Soybean Fatty Acid Desaturation Pathway:

soap making.

Responses to Temperature Changes and Pathogen Infection 115

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

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 warm-

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

temperature post-translational modulation of SACPD enzyme activity.

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

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 expression of desirable stress-acclimation traits.
