Accelerating gene function discovery by rapid phenotyping of fatty acid composition and oil content of single transgenic T₁ Arabidopsis and camelina seeds

Abstract

Arabidopsis is wildly used as a model plant and camelina is increasingly used for oilseed research and applications. Although the Arabidopsis genome has been sequenced for two decades, the functions of many lipid‐related genes and their regulators have not been well characterized. Improvements in the efficiency and accuracy of gene investigations are key to effective discovery of gene function and downstream bioengineering of plant oil quantity and quality. In this study, a visible marker was used to quickly identify transgenic T1 seeds and a method has been developed to phenotype fatty acid compositions and oil content of single T1 seeds. A whole seed direct transmethylation method was first optimized with multiple seeds and incubation at 85°C for 2 hours in a transmethylation solvent (5% H2SO4 in methanol with 30% toluene cosolvent) is recommended. Based on this method, a single Arabidopsis seed mini‐transmethylation (SAST) method has been established in a 1.5 ml GC sample vial with 200 μl transmethylation solvent. Characteristics of the method were evaluated and it was used to phenotype transgenic T1 seeds expressing AtFAD2 or RcWRI1. Our results indicate that fatty acid composition of T1 individual seeds are consistent with those of pools of multiple seeds from higher generations. However, oil content per individual seed varied substantially and therefore pooling five seeds is recommended for phenotyping oil content of T1 seeds. Additionally, a whole camelina single‐seed direct transmethylation was evaluated and results confirm its feasibility. The suitability of partial seed analysis of camelina was investigated but variation in composition of different seed tissues limits this approach.

Comprehensive and High-Coverage Lipidomic Analysis of Oilseeds Based on Ultrahigh-Performance Liquid Chromatography Coupled with Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry

“…Total lipids of rapeseed were extracted according to the procedure in previous reports, with minor modifications. ,, In short, crushed rapeseed (about 20 mg) was mixed with 5 mL of MeOH/CHCl 3 (1:2, v/v) and 10 μL of lipid internal standards. Subsequently, the mixture was vortexed at 1800 rpm for 1 h, followed by the addition of 1.33 mL of H 2 O to induce phase separation.…”

Concerted increases of FAE1 expression level and substrate availability improve and singularize the production of very‐long‐chain fatty acids in Arabidopsis seeds

“…The Pha:: FAE1 was transferred into Arabidopsis wild‐type Col‐0 and T 1 ‐transformed seeds were identified using red fluorescence. Because the fatty acid composition of a single Arabidopsis seed can be accurately determined by the SAST method (Ma et al., 2020), 39 T 1 seeds were phenotyped by GC analysis and the untransformed wild‐type Col‐0 single seeds were used as a control. The ratio of (20:1 + 22:1)/18:1 proportions was used to indicate the efficiency of substrate conversion to VLCFAs in these lines.…”

“…Mature T 1 seeds were collected and methyl‐esterified following the SAST method (Ma et al., 2020). Single seeds were treated with 200 µl methanol containing 5% (v/v) sulfuric acid, including 5 µl BHT plus 60 μl toluene and incubated at 85℃ for 2 hr in a 1.5 ml GC vial.…”

“…As for the T 3 homozygous seeds, 10 seeds were used in same process described above, and the injection volume of GC was 2 μl. The GC analysis method was described in our previous study (Ma et al., 2020).…”

Overexpression of the vacuolar sugar importer BvTST1 from sugar beet in Camelina improves seed properties and leads to altered root characteristics

“…Fatty acid analysis was performed as described earlier (Marmon et al, 2017) with the following modifications: Crushing the seeds before derivatisation was omitted (Ma et al, 2020), and for each plant, 3× 10–15 seeds were dried overnight at 105°C and incubated together with 150–250 μg of tripentadecanoin standard in 2 ml of a methanol/toluene (2:1, v/v) solution containing 2.5% (v/v) H 2 SO 4 (95%–97%) and 2% (v/v) dimethoxypropane at 80°C for 3 h. Given values are the trimmed means of individual plants after averaging the technical replicates for each plant and removing the highest and the lowest values per genotype.…”