Idesia polycarpa, Maxim, Maxim

Fan, Ruishen, Li, Long, Cai, Gui, Ye, Jing, Liu, Minhao, Wang, Shuhui & Li, Zhouqi, 2019, Molecular cloning and function analysis of FAD 2 gene in Idesia polycarpa, Phytochemistry 168, pp. 1-10 : 6-7

3. Discussion

Idesia polycarpa , a woody species with the potential for edible oil and biodiesel production, has a high quality and quantity of lipids and LA. The fruits of this species possess species-specific mechanisms for lipid accumulation. The relative ratio of fatty acids did not exhibit significant changes during the developmental process, both in the pericarp and the seed ( Li et al., 2016). These trends of fatty acid profiles are significantly different from the storage accumulation in other woody oil plants ( Dussert and Morcillo, 2013; Muñoz-Mérida et al., 2013; Galli et al., 2014). For example, in oil palm, dramatic changes in FA composition have been reported during the development of both the endosperm and the mesocarp ( Dussert and Morcillo, 2013). In this study, the accumulation of fruit lipids and LA contents differed among growth stages. Lipid accumulation was steady throughout the growth stages, being more rapid at 70–80 DAP and 100–110 DAP. The accumulation of LA occurred later than lipid accumulation, being more rapid at 80–90 DAP. This indicates that the synthesis of LA is regulated by the lipid content. These results may facilitate the selection of I. polycarpa fruits with various LA content requirements in different development stages.

Since the first plant FAD2 was cloned from Arabidopsis thaliana ( Nishiuchi et al., 1994) , more and more homologous FAD2 sequences have been cloned in plants. Based on previous studies, FAD2 is mainly responsible for the transformation of OA to LA in plants ( Heppard et al., 1996), but to date, FAD2 from I. polycarpa has not been cloned and characterised. In this study, we isolated FAD2 from I. polycarpa (IpFAD2) for the first time. Phylogenetic analysis indicated that IpFAD2 was closely related with PtFAD2 and PeFAD2. Introducing this gene into plants may help us to improve or reduce the LA content in I. polycarpa in the future.

The protein FAD2 belongs to the membrane- FADS -like superfamily, which contains membrane FADS s, alkane hydroxylases, beta carotene ketolases (CrtW-like), hydroxylases (CrtR-like) and other related proteins. They are present in all groups of organisms with the exception of archaea ( Xue et al., 2017). In the transcriptome of I. polycarpa pericarp and seed tissues, the metabolic enzymes associated with the biosynthesis of FA compositions in FAD genes are FAD2, FAD3, FAD5, FAD6, FAD7 and FAD8 ( Li et al., 2016). All of these genes belong to the membrane- FADS -like superfamily, in which FAD2 and FAD6 desaturate OA to LA. FAD3, FAD7 and FAD8 desaturate LA to ALA. Besides these desaturations, FAD5 encoding a palmitoyl-monogalactosyldiacylglycerol delta-7 desaturase, affects the accumulation of 16:3 by catalysing 16:0 MGDG to form 16:1 MGDG at position (Δ7) in leaves ( Wang et al., 2015; Hernández et al., 2005). Membrane FADS s are non-heme, iron-containing, oxygen-dependent enzymes involved in regioselective introduction of double bonds in fatty acyl aliphatic chains. They play an important role in the maintenance of the proper structure and functioning of biological membranes. This superfamily domain has extensive hydrophobic regions that would be capable of spanning the membrane bilayer at least twice ( Marchler-Bauer et al., 2017). Bioinformatics analysis of IpFAD2 suggested that it had a theoretical molecular mass of 44.03 kDa and an isoelectric point (pI) of 8.04. The secondary structure of IpFAD2 had five transmembrane helices and was constituted by alpha helix, extrend strand, beta turn and random coil. The IpFAD2 contained a membrane-FADS-like superfamily domain, located on the ER. This provides a reference for the LA synthesis theory of I. polycarpa in future studies.

Ectopic overexpression of the Perilla frutescens FAD 2 gene in Saccharomyces cerevisia could catalyse the conversion from OA to LA ( Xue et al., 2017), which is consistent with FAD2 enzyme activity in cotton ( Zhang et al., 2009), chia ( Xue et al., 2017), soybean ( Li et al., 2007) and olive ( Hernández et al., 2005). However, FAD2 from some plants such as Elaeis guineensis ( Sun et al., 2016) , Jatropha curcas ( Wu et al., 2013) , Aleurites fordii (Dyer JM et al., 2002) , Davidia involucrata ( Lei et al., 2010) , Perilla frutescens ( Lee et al., 2016) , camelina ( Rodríguez-Rodríguez et al., 2016) and Xanthoceras sorbifolia ( Guo et al., 2014) not only catalyses the conversion from OA to LA, can also produce hexadecadienoic acid (16:2 Δ9,12), with palmitoleic acid (16:1 Δ9) as catalysed substrate. This phenomenon might be associated with the preference of substrate or different transmembrane topologies of plant FAD2s ( Guo et al., 2014). In the present work, expression of IpFAD 2 in A. thaliana induce the conversion from OA to LA.

Similarly, qRT-PCR analysis was carried out to detect the relative transcript level of IpFAD2 at eight different growth stages of I. polycarpa fruits . Comparing the expression patterns and linoleic contents, we found that the IpFAD2 mRNA content was positively correlated with LA concentration in I. polycarpa fruits . When the IpFAD2 gene was exhibited at 80 DAP, the increase rate of LA was rapid in this stage, indicating that IpFAD2 plays a significant role in the accumulation and regulation of the PUFAs components, such as catalysing the conversion from OA to LA in fruits of I. polycarpa at different growth stages. Otherwise, the expression of IpFAD2 at 140 DAP was slightly higher than that 130 DAP (1.28425:1). In view of the low temperature after 130 DAP, this phenomenon can be explained by the fact that temperature drops lead to increased FAD2 expression of the cell ( Xue et al., 2017). These results can enrich our understanding that IpFAD2 not only regulates the change of LA, but also increases the cold resistance of I. polycarpa . At present, I. polycarpa is mainly planted in subtropical regions and difficult to cultivate in temperate and cold temperate regions. The results of our study can therefore facilitate the expansion of I. polycarpa cultivation.

In summary, we investigated the dynamic growth curve of lipid and LA accumulation in I. polycarpa fruits and analysed the increase rates of different growth stages. This study is the first report on full-length cDNA of IpFAD2 from I. polycarpa . This gene was expressed in different growth stages and had a positively relationship with the increase rate of LA in fruits. Bioinformatics analysis and prokaryotic expression demonstrated that IpFAD2 encodes a bio-functional ER-located-like FAD2 enzyme. The functional characterisation of this desaturase in A. thaliana confirmed that the isolated IpFAD2 proteins could catalyse LA synthesis. This study not only helps to understand the roles of IpFAD 2 in different growth stages, but also lays a foundation for the molecular regulation and coordination of high-LA traits of I. polycarpa . Further characterization of IpFAD2 and its functional relationship will provide more information about the mechanisms controlling FA composition in I. polycarpa , and possible ways to modify it such as genetic engineering breeding.