Comparison of sense and antisense methodologies for modifying the fatty acid composition of Arabidopsis thaliana oilseed

Cartea, María Elena; Migdał, Maciej; Galle, Anne-Marie; Pelletier, Guillaume; Guerche, Philippe

doi:10.1016/s0168-9452(98)00089-2

Cited by 40 publications

(15 citation statements)

References 27 publications

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“…The HP construct, which was targeted against the FAD2 coding region and had part of the FAD2 gene as the spacer region rather than a spliceable intron, was 25% less efficient than the iHP construct, but still produced a higher frequency of silencing than the CS construct. It is notable that the 66% efficiency of FAD2 silencing achieved with the CS construct in the current study is much higher than the 17% previously reported by Cartea et al (1998). This enhancement is possibly related to the different constructs used in the two studies.…”

Section: Discussioncontrasting

confidence: 78%

“…This enhancement is possibly related to the different constructs used in the two studies. The CS construct in the current work consisted of the rapeseed napin promoter (Fp1) driving a truncated FAD2 coding sequence that was missing the 5Ј-UTR and a small portion of the 5Ј coding region, whereas the construct used by Cartea et al (1998) consisted of the fulllength FAD2 coding sequence driven by rapeseed napin promoter. It is possible that this latter construct may result in expression of an effective ⌬12-desaturase in some transformants and, thereby, reduce the efficiency of silencing.…”

Section: Discussionmentioning

confidence: 99%

“…It is possible that this latter construct may result in expression of an effective ⌬12-desaturase in some transformants and, thereby, reduce the efficiency of silencing. In this regard, it is relevant that Cartea et al (1998) reported significantly higher efficiency of silencing (57%) using FAD2 antisense constructs. Conventional CS and antisense constructs often appear to induce PTGS by producing dsRNA when they are integrated into the genome in particular orientations, such as invertedrepeat arrangement.…”

Section: Discussionmentioning

confidence: 99%

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hpRNA-Mediated Targeting of the Arabidopsis FAD2 Gene Gives Highly Efficient and Stable Silencing

et al. 2002

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The endogenous ⌬12-desaturase gene (FAD2) in Arabidopsis was targeted for silencing using seed-specific cosuppression (CS), hairpin (HP) RNA (hpRNA), and intron-spliced HP (iHP) constructs. The iHP construct, incorporating the 120-bp 3Ј-untranslated region of the FAD2 gene, gave the highest degree of silencing. In some iHP lines ⌬12-desaturase activity was reduced to levels as low as those in the null fad2-1 mutant, and every primary transformant showed a pronounced reduction in FAD2 activity. One highly silenced iHP line was propagated for five generations and showed no reversion or diminution in its degree of silencing. About 75% of plants transformed with the HP construct, targeting the FAD2 coding region, gave dramatically reduced ⌬12-desaturase activity, whereas approximately 50% of plants transformed with the CS construct, containing the same coding region sequence, showed silencing at a much less profound level. In all three types of constructs, the degree of silencing was increased when the transgenes were homozygous, but this was much more pronounced for the CS constructs. All three types of construct could give a single locus that was capable of effective silencing, but in the one such CS line where this was the case, the locus had a complex insertion pattern. This is consistent with the concept that posttranscriptional gene silencing is induced by double-stranded, or self-complementary, RNA that is formed in cases of CS by complex insertion patterns at a single locus and that the most effective way of generating profoundly silenced plants is by the use of constructs that encode hpRNAs. Furthermore, these results demonstrate for the first time, to our knowledge, that iHP constructs targeted against an endogenous seed-expressed gene are clearly able to generate phenotypic changes that are inherited stably over several generations, making this approach a reliable technique for genetic modification of seed quality and possibly other traits in agricultural plants.

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Section: Discussioncontrasting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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hpRNA-Mediated Targeting of the Arabidopsis FAD2 Gene Gives Highly Efficient and Stable Silencing

et al. 2002

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“…A BglII restriction fragment of 7187 bp containing the PPR-A gene was subcloned into the unique BamHI site of the pEC2 binary vector (Cartea et al, 1998). A 7169-bp SpeI digestion fragment containing PPR-B was first subcloned into a modified pBluescript SK1 vector (Stratagene) containing two NotI sites and subsequently transferred into pEC2 at the NotI site.…”

Section: Plant Materialsmentioning

confidence: 99%

Characterization of Raphanus sativus Pentatricopeptide Repeat Proteins Encoded by the Fertility Restorer Locus for Ogura Cytoplasmic Male Sterility

et al. 2008

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Cytoplasmic male sterility is a maternally inherited trait in higher plants that prevents the production of functional pollen. Ogura cytoplasmic male sterility in radish (Raphanus sativus) is regulated by the orf138 mitochondrial locus. Male fertility can be restored when orf138 accumulation is suppressed by the nuclear Rfo locus, which consists of three genes putatively encoding highly similar pentatricopeptide repeat proteins (PPR-A, -B, and -C). We produced transgenic rapeseed (Brassica napus) plants separately expressing PPR-A and PPR-B and demonstrated that both encoded proteins accumulated preferentially in the anthers of young flower buds. Immunodetection of ORF138 showed that, unlike PPR-B, PPR-A had no effect on the synthesis of the sterility protein. Moreover, immunolocalization experiments indicated that complete elimination of ORF138 from the tapetum of anthers correlated with the restoration of fertility. Thus, the primary role of PPR-B in restoring fertility is to inhibit ORF138 synthesis in the tapetum of young anthers. In situ hybridization experiments confirmed, at the cellular level, that PPR-B has no effect on the accumulation of orf138 mRNA. Lastly, immunoprecipitation experiments demonstrated that PPR-B, but not PPR-A, is associated with the orf138 RNA in vivo, linking restoration activity with the ability to directly or indirectly interact with the orf138 RNA. Together, our data support a role for PPR-B in the translational regulation of orf138 mRNA.

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“…An 8.5-kb ClaI containing the entire SAH hydrolase 1 gene was subcloned into pBluescript KSþ. The plasmid was then linearized with NotI and ligated into pEC2 (Cartea et al, 1998). The resulting plasmid was transformed into Agrobacterium tumefaciens, and Arabidopsis thaliana transformation was performed using vacuum infiltration of flowers (Bechtold and Pelletier, 1998).…”

Section: Complementation Of the Hog1-1 Mutationmentioning

confidence: 99%

The Arabidopsis HOMOLOGY-DEPENDENT GENE SILENCING1 Gene Codes for an S-Adenosyl-l-Homocysteine Hydrolase Required for DNA Methylation-Dependent Gene Silencing

et al. 2005

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Genes introduced into higher plant genomes can become silent (gene silencing) and/or cause silencing of homologous genes at unlinked sites (homology-dependent gene silencing or HDG silencing). Mutations of the HOMOLOGY-DEPENDENT GENE SILENCING1 (HOG1) locus relieve transcriptional gene silencing and methylation-dependent HDG silencing and result in genome-wide demethylation. The hog1 mutant plants also grow slowly and have low fertility and reduced seed germination. Three independent mutants of HOG1 were each found to have point mutations at the 39 end of a gene coding for S-adenosyl-L-homocysteine (SAH) hydrolase, and hog1-1 plants show reduced SAH hydrolase activity. A transposon (hog1-4) and a T-DNA tag (hog1-5) in the HOG1 gene each behaved as zygotic embryo lethal mutants and could not be made homozygous. The results suggest that the homozygous hog1 point mutants are leaky and result in genome demethylation and poor growth and that homozygous insertion mutations result in zygotic lethality. Complementation of the hog1-1 point mutation with a T-DNA containing the gene coding for SAH hydrolase restored gene silencing, HDG silencing, DNA methylation, fast growth, and normal seed viability. The same T-DNA also complemented the zygotic embryo lethal phenotype of the hog1-4 tagged mutant. A model relating the HOG1 gene, DNA methylation, and methylation-dependent HDG silencing is presented.

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Comparison of sense and antisense methodologies for modifying the fatty acid composition of Arabidopsis thaliana oilseed

Cited by 40 publications

References 27 publications

hpRNA-Mediated Targeting of the Arabidopsis FAD2 Gene Gives Highly Efficient and Stable Silencing

hpRNA-Mediated Targeting of the Arabidopsis FAD2 Gene Gives Highly Efficient and Stable Silencing

Characterization of Raphanus sativus Pentatricopeptide Repeat Proteins Encoded by the Fertility Restorer Locus for Ogura Cytoplasmic Male Sterility

The Arabidopsis HOMOLOGY-DEPENDENT GENE SILENCING1 Gene Codes for an S-Adenosyl-l-Homocysteine Hydrolase Required for DNA Methylation-Dependent Gene Silencing

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