Increasing Oil Content in Cruciferae Seeds Through Modulating Seed Fatty Acid Reducer and Transparent Testa2
|Keywords||Brassica napus L. Arabidopsis SFAR TT2 BnLIP2 gibberellin signallingpathway DELLA protein fatty acid oil body seed germination seedling establishment leaf senescence|
Oilseed rape (Brassica napus L.) is one of the most important oil crops throughout the world. However, the mechanisms and the regulatory networks of seed oil accumulation are not clear. Arabidopsis, as a model plant species, and rapeseed belong to the same family, Cruciferae. In this study we investigated the function of GDSL-type SFAR (Seed Fatty Acid Reducer) and TT2(Transparent Testa2) in seed fatty acid metabolism, seed germination and seedling establishment in Arabidopsis, the effect of gibberellin (GA) signalling pathway on fatty acid metabolism and leaf senescence, and the relationship between the GA pathway and SFAR in fatty acid metabolism. We studied the effect of GA3and PAC on seed fatty acid content in oilseed rape through comparing the transcriptomes of Arabidopsis and brassica in response to exogenous application of GA3and PAC, and clarified the function of BnLIP2on seed fatty acid metabolism, seed germination and seedling establishment in Arabidopsis. These studies not only eludicate the molecular mechanisms of lipid metabolism, but also provide valuable information and gene resources for improving oil content and fatty acid procution in oil crops, such as oilseed rape, via either classical breeding or trangenic approaches.Our following results contribute to address the abovementioned questions:1. The effect of GDSL-type SFAR genes on fatty acid content and composition, and oil bodies in Arabidopsis seeds. We investigated the function of SFAR genes on seed fatty acid accumulation through analyzing the phenotypes of their T-DNA insertion mutants and overexpression transgenic plants. Our result showed that seeds of SFAR mutants contain more fatty acids, while those of their overexpression transgenic plants have less fatty acids, suggesting that SFAR genes are involved in degradation of fatty acids, and that they play redundant roles in regulating fatty acid metabolism. In addition, these SFAR genes affect the composition of fatty acids and oil body morphology in seeds.2. The relationship between the GA signalling pathway and SFAR genes. The "SFAR footprint" can be found in Arabidopsis mature seeds with enhanced GA signalling pathway. Meanwhile, several transcription factors that play essential roles in seed development and storage product accumulaton, several key genes involved in fatty acid biosynthesis, and the the five SFAR genes are significantly upregulated. In addition, seed oil bodies in Q-DELLA/gal-3mutants display similar morphology to those of SFAR overexpression transgenic plants. These results suggest that the GA pathway regulates the expression of SFAR genes downstream of DELLA proteins to affect fatty acid accumulation in Arabidopsis seeds.3. The effect of SFAR genes on seed germination and seedling establishment in Arabidopsis. On the MS medium containing6%glucose, the seed germination rate of SFAR overexpression plants is higher than that of wild type, and most of transgenic seedlings develop normal true leaves. On the other hand, PEG6000induces differential expression of many stress-related genes in35S::SFAR4seedlings. The "SFAR footprint" in35S::SFAR4may affect plant resistance to osmotic stresses.4. The effect of the GA pathway on leaf senescence and fatty acid metabolism in Arabidopsis. The relevant parameters, including leaf colour, dry weight, chlorophyll, total soluble sugar, and leaf fatty acid content and compositions change significantly during leaf senescence in DELLA mutant (Q-DELLA/gal-3and gal-3). Expression of SAG12and SAG29, some genes involved in the (3-oxidation pathway and in several phytohormone pathways is affected significantly. Thus, the GA signaling pathway may interact with other phytohormones through DELLA proteins to promote leaf senescence in Arabidopsis.5. The effect of the GA pathway on seed fatty acid content in oilseed rape. Exogenous application of500μM GA3results in an increase in seed dry weight, but a decrease in fatty acid content in Zheshuang72and Zheshuang758. This is consistent with the result in Arabidopsis. However,500μM PAC treatment had a similar effect on seed dry weight and seed fatty acid content to GA treatment in these two rapeseed genotypes, indicating that the concentration of PAC may be stressful to rapeseed growth and development and thus represses seed fatty acid accumulation.6. The effect of BnLIP2on seed fatty acid accumulation, seed germination and seedling establishment in Arabidopsis. In developing seeds of Zheshuang72and Zheshuang758, BnLIP2expression gradually decreases. Exogenous application of GA3induces BnLIP2expression. Although overexpression of BnLIP2has no effect on seed fatty acid accumulation, it significantly increases plant resistance to osmotic stress during seed germination and seedling establishment. These results suggest that the GA pathway may affect fatty acid content through other GDSL-type genes or other pathways rather than BnLIP2in oilseed rape.7. The effect of TT2on seed fatty acid accumulation and resistance of young seedlings to abiotic stresses in Arabidopsis and their underling molecular mechanisms. We found that loss of function of TT2results in a decrease in seed size and seed weight, an increase in seed fatty acid content and the change in seed fatty acid composition. These phenotypes could be due to the following reasons:1) the lighter seed coat contributes to an increase in the relative embryo size and weight;2) more AcCoA flows into the fatty acid biosynthetic pathway;3) FUS3and some key genes in the fatty acid biosynthetic pathway are upregulated;4) decreased seed total and storage protein content. On the other hand, tt2mutants are more sensitive to abiotic stresses (higher concentrations of NaCl, Glucose and ABA) during seed germination and seedling establishment in Arabidopsis.Therefore, when we manipulate BnSFAR and BnTT2to increase rapeseed oil content, we should take into consideration their effects on plant respones to abiotic stresses. It is also practially feasible to perform PAC treatment to affect GA signaling, thus increasing rapeseed oil content. The actual application of this approach will need further opimization of PAC concentration and identification of appropriate developmental stages of rapeseed at which PAC treatment could be effective.