FAD2 Cosuppression,a Bottleneck For Increasing Polyunsaturated Fatty Acid Content In Oilseeds,and Investiigation Of Its Mechanism

In mammals,including human,there is no fatty acid desaturase converting monounsaturated fatty acids to polyunsaturated fatty acids（PUFA）,which is essential and must be taken from diets.Triacylglycerol（TAG）is one of the most important storage components of seed oil.In seeds,phosphatidylcholines（PCs）,which are the substrates for fatty acid desaturation,are catalyzed in endoplasmic reticulum by fatty acid desaturase 2（FAD2）and FAD3 to generate 18:2 and 18:3,respectively.In order to increase PUFA content in oilseeds,TmFAD2 and AtFAD2 were introduced into flax and camelina.Combined with the previous researches on FAD2 transgene and the results presented here,FAD2cosuppression was suggested as the bottleneck to increasing PUFA contents in oilseeds.To further study the mechanism and breakthrough the bottleneck in FAD2 bioengeering,AtFAD2 or mutated FAD2s were transformed into Arabidopsis wild type and mutants.Based on the studies on FAD2 cosuppression,the results are as follows:1,Strong FAD2 cosuppression impedes increase of PUFA content in oilseeds（A）Seed-specific expression of TmFAD2 and AtFAD2 in flax and camelina both resulted in decreased PUFA contents.Combined with further analysis on T₃ seeds of Brassica carinata transgenic lines with Bc FAD2,FAD2 transgenes trigger the decrease of PUFA contents in seeds,which may be the conversed behaviors in oilcrops and the bottleneck in FAD2 bioengineering.（B）The results of PUFA content and FAD2 expression level in Arabidopsis transgenic lines indicate that AtFAD2 transgene triggered strong gene cosuppression in 80%lines,and decreases of PUFA contents in severe lines were as same as that in fad2-1 mutant.In cosupression lines,21-22 nt si RNAs derived from FAD2 coding region increased 200-300times,comparing with that in wild type.Moreover,a phenomenon of transgenerational instability was detected in advancing generations of Pha::AtFAD2/Col-0 lines.（C）In Arabidopsis rdr6 mutant,FAD2 was effectively overexpressed and increased seed PUFA contents up to 63.9%and 64%from 52%in background.Besides,the increased PUFA contents were stable in advancing generations of Pha::AtFAD2/rdr6 lines.2,Silencing pathway of FAD2 cosuppression（A）The cosuppression frequencies in T₁ seeds of mutants indicated that DCL2 and DCL4 functioned redundantly in FAD2 cosuppression.FAD2 cosuppression was relieved in85%transgenic lines of dcl2 dcl4 double mutant,but not in transgenic lines of either dcl2 or dcl4 single mutants.FAD2 cosuppression was also relieved in ago1-25 mutant.（B）DCL1 and MOS2 were also suggested to meidate FAD2 cosuppression.FAD2cosuppression frequency decreased to 23.5%and 0%in dcl1-9 and mos2-2 mutants,respectively.It was noting that no antisense si RNAs derived from AtFAD2 were detected in Pha::AtFAD2/dcl1-9 and Pha::AtFAD2/mos2 lines.3,The trigger of FAD2 cosuppression（A）By micro RNA target prediction,miR2938 was deduced as the most possible micro RNA to target AtFAD2.However,FAD2 mutation for increasing miR2938 mismatch level did not relive gene cosuppression.Based on the further analysis on antisense transcripts of FAD2,a putative stem-loop structure was presumed to generate micro RNA-like small RNAs.Similarly,FAD2 mutations on the sencodary structure were suggested to be unrelated to FAD2 cosuppression.Thus,micro RNAs may be not the triggers of FAD2 cosuppression.（B）The native terminator of AtFAD2 and the 153 bp truncation 5′end had no effect on the frequencies of FAD2 cosuppression.On the contrary,352 bp truncation of 3′end was found to decrease FAD2 cosuppression frequency to 65%,suggesting that the 3′region（352bp）of FAD2 may be involved in FAD2 cosuppresion.4,Characteristics of ab RNA in FAD2 cosuppression and an RDR6-independent cosuppression pathway（A）In this study,FAD2 cosuppression level was enhanced in both ein5-1 and ski2-3mutants,suggesting that both 5′-and 3′-end aberrant RNAs are s involved in the process of FAD2 cosuppression.（B）Further investigation of FAD2 cosuppression in rdr6 ein5 ski2 triple mutant indicated that weak FAD2 cosuppression still existed in the rdr6 ein5 ski2 triple mutant.It is suggested for the first time that an RDR6-independent transgenic gene silencing was observed in plants,which is activated or enhanced under dysfunction of both the RNA decay system and RDR6.5,Transgenerational instability in Pha::AtFAD2/rdr2 linesIn T₁ generation of Pha::AtFAD2/rdr2 lines,FAD2 cosuppression was enhanced,comparing with that in AtFAD2 transgenic lines of Col-0.But in T₂ lines and T₃ lines of Pha::AtFAD2/rdr2,FAD2 cosuppression frequencies were 50%and 20%,respectively.These results suggested that the epigenetic modifications may impede the gene cosuppression in the absent of RDR2.