Final Report Summary - PIF OIL (Regulation of Seed Storage Oil Mobilization (SOM) by the Phytochrome-Interacting bHLH Transcription Factors PIFs)
In seed, lipid is found mainly in triacylglycerol (TAG) form and is stored in small spherical organelles called lipid bodies (LBs).Right after germinating, TAGs are hydrolyzed by lipase(s) and metabolized into carbon through the beta-oxydation pathway and glyoxylate cycle inside the glyoxysome. During the first days following the germination, seedling growth fully relies on the sugar released from breakdown of lipid accumulated in seeds until the seedling becomes photosynthetic and autotrophic
In laboratory, we are working on a family of bHLH transcription factors called PIFs for phytochrome Interacting factors (six in Arabidopsis: PIF1, 3, 4, 5, 6 and 7). In the dark, PIFs accumulate and act as repressors of seedling deetiolation process. Upon seedling exposure to light, phytochrome (phy) phototoreceptors are activated and translocated to the nucleus where they interact with the PIFs and induce their rapid degradation. This light-induced degradation reverses the PIF repressing action in the dark and allows the initiation of photomorphogenesis. A quadruple mutant deficient in PIF1, PIF3, PIF4 and PIF5 (termed as pifq) displays morphogenic development in total darkness that strongly phenocopies that of normal light-grown seedlings (Figure 1). Microarray expression analysis of pifq reveals that most of enzymes implicate in lipid mobilization pathway are deregulated in pifq in dark. Comparison of LBs content in cotyledons by microscopic examination reveals a significant reduction in lipid bodies’ number per cell in the pifq mutant, a phenotype similar to what we observed in col-0 grown in light (Figure 2). The aim of the project is to understand how light impacts lipid metabolism during seedling development and how PIFs are involved in this regulation.
Light regulation of lipid mobilization was analyzed by two approaches. First, we used the Nile Red dye to stain in vivo, lipid bodies in cotyledons. Reduction of LBs upon time was observed in col-0 and pifq grown in light and total dark. In parallel, disappearance of TAG right after germination was quantified by Gas Chromatography-Mass Spectrometry to evaluate rate of lipid mobilization between genotype and light conditions. This part was done in collaboration with two European partners, the well-known institutes of CNRS in France and CSIC in Spain. Here, by using real-time quantitative PCR, we analyzed expression of master gene list that include proteins involved in oilseed breakdown pathway such as lipases, beta oxidation and glyoxylate cycle enzymes. To define the gene regulate by PIFs, expression was measured in developing seedling (from 1 to 4 days) of col-0 and pifq grown in light and total dark conditions. Using chromatin immunoprecipitation assay, we plan to identify which genes are PIF-bound targets and start a functional analysis on the selected candidates.
This multidisciplinary project combines physiologic, metabolic and molecular approaches and benefit from the expertise in plant lipid of various European laboratories.Efficient storage oil breakdowns is essential for successful seedling establishment, which in turn is of paramount importance for plant fitness in the field. Knowing the molecular mechanisms that control this process might lead to important applications in plant biotechnologies as plant-derived oils are essential for both food and feed applications.
In laboratory, we are working on a family of bHLH transcription factors called PIFs for phytochrome Interacting factors (six in Arabidopsis: PIF1, 3, 4, 5, 6 and 7). In the dark, PIFs accumulate and act as repressors of seedling deetiolation process. Upon seedling exposure to light, phytochrome (phy) phototoreceptors are activated and translocated to the nucleus where they interact with the PIFs and induce their rapid degradation. This light-induced degradation reverses the PIF repressing action in the dark and allows the initiation of photomorphogenesis. A quadruple mutant deficient in PIF1, PIF3, PIF4 and PIF5 (termed as pifq) displays morphogenic development in total darkness that strongly phenocopies that of normal light-grown seedlings (Figure 1). Microarray expression analysis of pifq reveals that most of enzymes implicate in lipid mobilization pathway are deregulated in pifq in dark. Comparison of LBs content in cotyledons by microscopic examination reveals a significant reduction in lipid bodies’ number per cell in the pifq mutant, a phenotype similar to what we observed in col-0 grown in light (Figure 2). The aim of the project is to understand how light impacts lipid metabolism during seedling development and how PIFs are involved in this regulation.
Light regulation of lipid mobilization was analyzed by two approaches. First, we used the Nile Red dye to stain in vivo, lipid bodies in cotyledons. Reduction of LBs upon time was observed in col-0 and pifq grown in light and total dark. In parallel, disappearance of TAG right after germination was quantified by Gas Chromatography-Mass Spectrometry to evaluate rate of lipid mobilization between genotype and light conditions. This part was done in collaboration with two European partners, the well-known institutes of CNRS in France and CSIC in Spain. Here, by using real-time quantitative PCR, we analyzed expression of master gene list that include proteins involved in oilseed breakdown pathway such as lipases, beta oxidation and glyoxylate cycle enzymes. To define the gene regulate by PIFs, expression was measured in developing seedling (from 1 to 4 days) of col-0 and pifq grown in light and total dark conditions. Using chromatin immunoprecipitation assay, we plan to identify which genes are PIF-bound targets and start a functional analysis on the selected candidates.
This multidisciplinary project combines physiologic, metabolic and molecular approaches and benefit from the expertise in plant lipid of various European laboratories.Efficient storage oil breakdowns is essential for successful seedling establishment, which in turn is of paramount importance for plant fitness in the field. Knowing the molecular mechanisms that control this process might lead to important applications in plant biotechnologies as plant-derived oils are essential for both food and feed applications.
