Periodic Reporting for period 1 - PlaBioC (FORMATION OF HIGH-VALUE DENSE PLANT BIOCONDENSATES)
Reporting period: 2022-12-01 to 2024-11-30
To achieve a more sustainable future, we must transition from fossil fuel-based production systems to greener alternatives. Instead of relying on carbon-emitting processes, we need to embrace bioproduction, where carbon dioxide serves as the sole carbon source. In nature, photosynthetic organisms master this process. However, expanding this environmentally friendly system to synthesize high-value compounds that are either not produced or only available in low amounts in current photosynthetic systems presents a challenge.
A critical aspect of bioproduction is the ability to store high-value natural products without causing toxicity to the host organism. By studying plants that naturally accumulate these compounds in high concentrations; we aim to transfer the knowledge to engineered bioproduction systems.
The PlaBioC project focused on two model plants known for their ability to store natural products in high amounts: Vanilla planifolia, which accumulates vanillin glucoside at a concentration of 2.2 kg/L in the inner part of maturing pods and Sorghum bicolor, which produces the defence compound dhurrin, reaching up to 30% of dry mass in the upper tissues of seedlings. The primary goal was to determine whether these plants store these compounds in dense biocondensates and to identify key factors involved in their formation.
A critical aspect of bioproduction is the ability to store high-value natural products without causing toxicity to the host organism. By studying plants that naturally accumulate these compounds in high concentrations; we aim to transfer the knowledge to engineered bioproduction systems.
The PlaBioC project focused on two model plants known for their ability to store natural products in high amounts: Vanilla planifolia, which accumulates vanillin glucoside at a concentration of 2.2 kg/L in the inner part of maturing pods and Sorghum bicolor, which produces the defence compound dhurrin, reaching up to 30% of dry mass in the upper tissues of seedlings. The primary goal was to determine whether these plants store these compounds in dense biocondensates and to identify key factors involved in their formation.
To investigate vanillin glucoside accumulation in Vanilla planifolia, pod samples at various developmental stages after pollination were sectioned and analyzed using analytical chemistry techniques. This approach enabled us to track the vanillin glucoside accumulation over time and select optimal time points for gene expression analysis through transcriptomics. As a result, we identified a list of co-expressed genes that follow the same accumulation pattern as vanillin glucoside, increasing in concentration in the inner pod tissue over time but absent in leaf tissue.
For Sorghum bicolor, we developed a method to isolate and separate different cell types from seedling leaves. Cell isolation techniques were combined with flow cytometry, using size and chlorophyll autofluorescence as a sorting parameter to distinguish between epidermal and mesophyll cells. This approach successfully yielded two distinct cell populations for further chemical and proteomic analysis. Our findings confirmed that dhurrin is predominantly stored in epidermal cells, but the proteins involved in its metabolism did not exhibit a clear distribution pattern, suggesting the involvement of alternative transport mechanisms. Additionally, we optimized cell isolation protocols to enable transient transformation with fluorescently tagged dhurrin-related proteins, allowing us to determine their subcellular localization through microscopy.
For Sorghum bicolor, we developed a method to isolate and separate different cell types from seedling leaves. Cell isolation techniques were combined with flow cytometry, using size and chlorophyll autofluorescence as a sorting parameter to distinguish between epidermal and mesophyll cells. This approach successfully yielded two distinct cell populations for further chemical and proteomic analysis. Our findings confirmed that dhurrin is predominantly stored in epidermal cells, but the proteins involved in its metabolism did not exhibit a clear distribution pattern, suggesting the involvement of alternative transport mechanisms. Additionally, we optimized cell isolation protocols to enable transient transformation with fluorescently tagged dhurrin-related proteins, allowing us to determine their subcellular localization through microscopy.
The project generated several valuable datasets relevant to researchers working with Vanilla planifolia and Sorghum bicolor. These include:
• Quantitative data on vanillin glucoside accumulation in different pod tissues and developmental stages.
• A transcriptomic dataset identifying genes co-expressed with vanillin glucoside accumulation.
• Optimized methodologies for transient cell transformation and tissue-specific cell separation in Sorghum.
By linking vanillin accumulation data with transcriptomic analysis, we compiled a list of candidate genes potentially involved in vanillin storage and biocondensate formation. Future research is needed to confirm their precise roles. Similarly, our insights into dhurrin storage and transport bring us closer to understanding its compartmentalization mechanisms, although additional studies are required to confirm the presence of biocondensates.
Overall, the PlaBioC project advances our understanding of metabolite storage in plants and provides valuable tools for researchers interested in engineering sustainable bioproduction systems.
• Quantitative data on vanillin glucoside accumulation in different pod tissues and developmental stages.
• A transcriptomic dataset identifying genes co-expressed with vanillin glucoside accumulation.
• Optimized methodologies for transient cell transformation and tissue-specific cell separation in Sorghum.
By linking vanillin accumulation data with transcriptomic analysis, we compiled a list of candidate genes potentially involved in vanillin storage and biocondensate formation. Future research is needed to confirm their precise roles. Similarly, our insights into dhurrin storage and transport bring us closer to understanding its compartmentalization mechanisms, although additional studies are required to confirm the presence of biocondensates.
Overall, the PlaBioC project advances our understanding of metabolite storage in plants and provides valuable tools for researchers interested in engineering sustainable bioproduction systems.