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Transcriptional regulation of oil accumulation in the endosperm of oilseeds

Final Report Summary - TRIANON (Transcriptional regulation of oil accumulation in the endosperm of oilseeds)

- Summary description of the project objectives:
Consumption of oil derived from seeds is steeply increasing worldwide. First, oilseeds represent a key component of human and livestock diets. Then, fatty acids found in storage lipids accumulated in oilseeds are structurally similar to long chain hydrocarbons, so that plant oils are now widely used as competitive and sustainable alternatives to hydrocarbon-based products for energy and green chemistry (synthesis of detergents, plastics, lubricants, and paints). The increasing demand of plant oils for both nutritional and industrial applications highlights the urgent need to develop original methodologies allowing the increase of seed oil content when limited progress has been obtained by conventional breeding over the last decade. Elucidation of the molecular mechanisms controlling oil storage in seeds will ultimately allow engineering domesticated high yielding oil crop species. In oilseeds, two compartments, namely the endosperm and the embryo, can accumulate oil to different extents, depending on the species considered. Although recent advances have contributed to the elucidation of the transcriptional regulation of oil storage within embryo structures, our understanding of the regulatory processes controlling oil accumulation within the endosperm remains very limited. The TRIANON project was set up to elucidate the transcriptional regulation of oil accumulation in the endosperm in the model plant Arabidopsis thaliana, an oleaginous species of the Brassicaceae family.
Three key research objectives were attained:
1. Isolating new transcriptional regulators participating in the control of oil storage within the endosperm of oilseeds.
2. Elucidating the molecular mechanism involving MYB115 and MYB118, two of these transcriptional regulators.
3. Exploiting this knowledge to modify oil accumulation in a tissue-specific manner (in seeds) in the frame of a biotechnological approach.

- Description of the work performed:
Implementation of various complementary strategies exploiting available transcriptomic resources and collections of insertion mutants have led to the isolation of several transcriptional regulators controlling oil metabolism in the endosperm of maturing seeds in the model higher plant A. thaliana. Some of these transcription factors were already known for their regulatory action over oil metabolism in other seed compartments (e.g. the embryo) whereas others, specifically expressed in the endosperm, had never been related before to the control of this metabolism.
Two of these transcriptional regulators, namely MYB115 and MYB118, were thoroughly characterized. The expression patterns of these MYBs as well as their transcriptional activation were first characterized. Identification of target genes of these transcription factors was then carried out. The two closely related transcription factors share target genes and their functional redundancy was further established by the study of simple and double mutants affected in the corresponding MYB115 and MYB118 genes. In mutant seeds, both the quantity of oil stored in the endosperm and the fatty acid composition of this oil were affected. Positively regulated genes of the transcription factors encoding palmitoyl-ACP desaturases were finally characterized to elucidate the molecular bases of omega-7 fatty acid synthesis, these monoenoic fatty acid species being mostly accumulated in the endosperm of A. thaliana seeds.
Finally, constructs aimed at modifying oil accumulation in seeds were prepared that exploited the knowledge generated within the frame of this research program. They were assayed in A. thaliana as a proof of concept. A first strategy aimed at increasing the endosperm oil content (at the expense of the embryo) was tested. Then, a set of constructs aimed at increasing the seed omega-7 content was prepared and tested.

- Description of the main results achieved:
In the model higher plant A. thaliana, seed filling is characterized by the storage of oil during the maturation process. This oil is partitioned between the embryo and the endosperm. The pattern of deposition, final amount, and fatty acid composition of these reserves differ between the two compartments, with the embryo representing the main storage site. Interestingly, endosperm oil is enriched in omega-7 monoenes, that represent 25 Mol% of total FAs, whereas these molecular species are proportionally less abundant in the embryo, only accounting for 3 Mol%. Complementary approaches have shown the importance of master transcriptional regulators of the maturation process (e.g. LEC1, LEC2, ABI3) for the developmentally controlled activation of oil biosynthesis in the two zygotic tissues of the seed. As for the MYB118 transcription factor, transcriptionally induced at the onset of the maturation phase in the endosperm, it counteracts the action of the master regulators in this tissue, thus promoting a differential partitioning of reserves between the embryo and the endosperm. MYB115, a close homolog of MYB118, is also induced in the maturing endosperm. The two MYB transcription factors share a set of transcriptional targets and exhibit partially redundant functions. In the endosperm of the myb115 myb118 double mutant, oil content is doubled compared to that of the wild type, while omega-7 fatty acids disappear. The identification of two genes encoding palmitoyl-ACP desaturases (PADs) that are directly activated by MYB115 and MYB118 in the endosperm allowed us to reconcile these apparent discrepancies. While repressing the overall rate of oil biosynthesis in the endosperm, the two transcription factors activate two PADs that enhance the proportion of omega-7 in this oil. An initial characterization of the structure-function relationship for these enzymes revealed residues lining their substrate pocket that are critical for substrate specificity and that distinguish them from other acyl-ACP desaturases encoded by the A. thaliana genome (e.g. FAB2).

- Final results and their potential impact and use:
The main outcome of the project consists in original knowledge about the regulation of fatty acid production in plants. The results of the project are mainly fundamental, even though biotechnological tools have been developed in the model species A. thaliana as proof of concept. In a near future, strategies tested within the frame of this project may be applied to the improvement of oil yields and to the modification of fatty acid composition in oilcrop species such as Brassica napus or Camelina sativa and the generation of new varieties of bioeconomic interest.