Strong co-suppression impedes an increase in polyunsaturated fatty acids in seeds overexpressing <i>FAD2</i>

Du, Can; Chen, Yangyang; Wang, Kai; Zheng, Yanan; Zhao, Conghai; Jia, Qingli; Taylor, David C.; Zhang, Meng

doi:10.1093/jxb/ery378

Cited by 28 publications

(27 citation statements)

References 40 publications

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“…Results showed that 18:1 increased in 23 out 30 T 1 seeds compared with untransformed seeds and most lines showed an absolute 20%-40% net increase of 18:1 compared with untransformed seeds (e.g., 18:1 increased from 15% to 35% and to 55%.). This nearly 80% frequency of co-suppression and the intensity of co-suppression closely matched the previous results for T 2 seeds (Figure 5b in this study and Figure 2b of Du et al, 2018). These results indicate that determining fatty acid composition of T 1 seeds by the SAST method can phenotype transgenic lines early and reliably.…”

Section: Application Of the Sast Or Modified Sast To Phenotype Fattsupporting

confidence: 91%

“…Analysis of larger numbers of independent lines also allows for more robust statistical analysis. For example, overexpressing sense AtFAD2 triggers a strong co-suppression with a high frequency, which was clearly indicated with statistical confidence by analysis of dozens of transgenic lines (Du et al, 2018). In the present study, dozens of T 1 seeds from the same vector transformation were collected and individual T 1 seeds were used for phenotyping their fatty acid compositions.…”

Section: Advantages Of Sast Methodssupporting

confidence: 53%

“…In our previous studies, overexpression of sense FAD2 triggers a strong co-suppression and the substrate of FAD2 (18:1) is accumulated at a high frequency in seeds of flax, carinata, camelina, and Arabidopsis (Du et al, 2018). To characterize this strong co-suppression, we previously determined fatty acid profiles of pooled transgenic Arabidopsis seeds in dozens of independent T 2 and higher generation lines (Du et al, 2018). This same phenotype was used in the present study to test the changes of fatty acid composition on individual T 1 seeds using the SAST method.…”

Section: Application Of the Sast Or Modified Sast To Phenotype Fattmentioning

confidence: 89%

“…For example, a decrease of the expression of the resident hygromycin resistance was found in T 4 generation (Ondřej et al, 1999) and expressing BcFAD2 leads to a difference in average fatty acid proportions in transgenic T 3 and T 4 seeds compared to T 2 generations (Du et al, 2018);…”

Section: Advantages Of Sast Methodsmentioning

confidence: 99%

“…In order to test the validity of the SAST or modified SAST for this early identification, we characterized the phenotypes of transgenic lines in their T 1 seeds. In our previous studies, overexpression of sense FAD2 triggers a strong co-suppression and the substrate of FAD2 (18:1) is accumulated at a high frequency in seeds of flax, carinata, camelina, and Arabidopsis (Du et al, 2018). To characterize this strong co-suppression, we previously determined fatty acid profiles of pooled transgenic Arabidopsis seeds in dozens of independent T 2 and higher generation lines (Du et al, 2018).…”

Section: Application Of the Sast Or Modified Sast To Phenotype Fattmentioning

confidence: 99%

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

Ohlrogge

et al. 2020

Plant Direct

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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.

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Section: Application Of the Sast Or Modified Sast To Phenotype Fattsupporting

confidence: 91%

Section: Advantages Of Sast Methodssupporting

confidence: 53%

Section: Application Of the Sast Or Modified Sast To Phenotype Fattmentioning

confidence: 89%

Section: Advantages Of Sast Methodsmentioning

confidence: 99%

Section: Application Of the Sast Or Modified Sast To Phenotype Fattmentioning

confidence: 99%

See 3 more Smart Citations

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

Ohlrogge

et al. 2020

Plant Direct

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

Taylor

et al. 2021

Plant Direct

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Our initial goal was to evaluate the contributions of high 18:1 phosphatidylcholine and the expression level of FAE1 to the accumulation of very‐long‐chain fatty acids (VLCFAs), which have wide applications as industrial feedstocks. Unexpectedly, VLCFAs were not improved by increasing the proportions of 18:1 in fad2‐1 mutant, FAD2 artificial miRNA, and FAD2 co‐suppression lines. Expressing Arabidopsis FAE1 resulted in co‐suppression in 90% of transgenic lines, which was effectively released when it was expressed in the rdr6‐11 mutant host. When FAE1 could be highly expressed, apart from its naturally preferred product, 20:1, other saturated and polyunsaturated VLCFAs also accumulated in seeds. We postulated that overabundant FAE1 might cause the diversified VLCFA profile. When FAE1 was highly expressed, knocking down FAD2 increased the content of 20:1, suggesting that the 18:1 availability in the acyl‐CoA pool increased from the high 18:1‐PC via acyl editing. Concurrent decreases of side products like 22:1 and 20:0 in these lines suggest that increasing availability of the preferred substrate could suppress the side elongation reactions and reverse the effect of VLCFA product diversification due to overabundant FAE1. Re‐analysis of FAD2 knockdown lines indicated that increasing 18:1 led to a decrease of 22:1, which also supports the above hypothesis. These results demonstrate that 18:1 substrate could be increased by a downregulation of FAD2 and that a balance between the levels of enzyme and substrate may be crucial for engineering‐specific VLCFA products.

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How the xerophytic moss Pogonatum inflexum tolerates desiccation

Liu,

Zhang,

et al. 2024

Plant Cell Rep

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Strong co-suppression impedes an increase in polyunsaturated fatty acids in seeds overexpressing FAD2

Cited by 28 publications

References 40 publications

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

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

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

How the xerophytic moss Pogonatum inflexum tolerates desiccation

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Strong co-suppression impedes an increase in polyunsaturated fatty acids in seeds overexpressing FAD2

Cited by 28 publications

References 40 publications

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

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

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

How the xerophytic moss Pogonatum inflexum tolerates desiccation

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

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