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Maturation of plant autophagosomes: mapping the route to sustainability

Periodic Reporting for period 1 - MAPoPHAGY (Maturation of plant autophagosomes: mapping the route to sustainability)

Reporting period: 2018-09-21 to 2020-09-20

Upcycling of cellular content is a crucial mechanism allowing cells to remove damaged or superfluous content and also produce energy and building blocks in the absence of external resources. In the cells of animal, fungi and plants this is done by a molecular pathway called autophagy (Greek for “self-eating”). Autophagy is generally conserved in all three eukaryotic kingdoms of life, however the mechanism has also acquired kingdom-specific mechanisms.
Plant autophagy plays a crucial role in crops fitness, biomass and seed yield, tolerance to changing climate and pathogens. Thus thorough understanding of this mechanism is extremely important for improving crop performance.
Originally autophagy was best understood using fungal cells as a model organism. Consequently, the importance of autophagy for human health promoted extensive research on its molecular mechanisms in animal cells. Plant autophagy research has greatly progressed in the past decade, but still heavily relies on extrapolation of the knowledge obtained on animal and yeast model systems.
In my project I performed fundamental research focused on investigating plant-specific aspects of autophagy that are essential for understanding the regulation and function of the pathway in plants. To enable my studies I established assays that will be of general use for plant-related research.
Activation of the autophagic pathway in cells leads to formation of double membrane vesicles, autophagosomes, that sequester the cargo destined for degradation and deliver it to the lytic compartment where it is processed and recycled. The molecular mechanisms governing of autophagosome formation, maturation, trafficking to the lytic compartment (lytic vacuoles in plants) are complex and well-orchestrated with other processes ongoing in the cell.
The main findings of this project describe new intriguing insights on the plant-specific aspects of autophagosomes maturation:
• roles of tethering complexes
• post-translational modifications of the essential autophagy-related protein ATG8
• cross-talk between autophagy machinery and biogenesis of the lytic vacuole, the final destination of the autophagosomes
Additionally, during my project I established a number of devoted protocols that are already being implemented by other research groups and established a set of transgenic lines, which will be of a great use for autophagy-related plant research. The main results are partially available on my website and are being prepared for the publication in a form of four manuscripts.
The fundamental findings made within this project provided an important insight into plant-specific aspects of autophagy, its regulation and function. Currently I am working on investigating in more detail the molecular mechanisms underpinning my observations.
To enable the studies planned for my project I established RoPod (https://www.alyonaminina.org/ropod) a system for advanced fluorescent microscopy optimized for Arabidopsis roots with the aim to enable long-term time-lapse imaging and reduce mechanical stress typically cause by such assays.
Additionally, I developed a 3D printed robot for low-cost high-throughput automated imaging of Petri plates (Smart Plate Imaging Robot, SPIRO, https://www.alyonaminina.org/spiro). This imaging platform is supplemented with the web-based graphical user interface allowing remote control of the experiments, which became and extremely useful feature allowing me to perform experiments even during pandemic-triggered lockdown of the host laboratory. Furthermore, we developed two most typical phenotyping assays for plant seedlings that are based on images acquired by SPIRO: seed germination and root growth assays. SPIRO is mostly 3D printed, it is open source and extremely cheap compared to commercially available solutions. It also has a small footprint and fits into typical plant growth cabinets enabling continuous imaging under favourable conditions. This platform will be of general use for plant research community.