Autophagy, Proteases and plant PErformaNces – hAPPEN

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The approach chosen for the mechanistic and physiological study of autophagy and associated proteases is a reverse genetics approach using plants in which the functions of autophagy and proteases are abolished by mutation or stimulated by genetic modification.

The phenotypes of these inhibited or stimulated plants are compared to those of the wild-type plant, which serves as a reference. The phenotypes studied are located at various scales. At the whole-plant level, nitrogen flux analyses are performed using isotopic labeling, biomasses are measured, and the level of leaf senescence is assessed during development.

Microscopy imaging makes it possible to assess the structural modifications caused by defects or excesses of autophagy and proteases. It also allows visualization of autophagic activity by counting autophagic vesicles.

Changes in protein content are assessed by proteomic analyses, impacts on metabolism by metabolomic approaches.

The hAPPEN project showed that the different proteases induced during senescence and suspected for their roles in nutritional recycling were not individually essential, because they were probably redundant. When one of them is absent, its deficiency can be compensated by another. Conversely, autophagy is absolutely essential for the recycling and remobilization of nitrogen to the seed, and autophagy mutants show a remobilization deficit that can be up to a 60% reduction compared to the wild-type plant. The project also showed that stimulating autophagy significantly increased the flow of remobilized nitrogen to the seed and made it possible to obtain an increase in the protein level in the seed. These results therefore show that cellular autophagy is a lever for increasing nitrogen recycling, which is favorable to the reduction of nitrogen inputs. The hAPPEN project also demonstrated that protein resources in plant roots are significant for nitrogen remobilization to the grain. This project made it possible to dissect and characterize the metabolic events occurring in roots as they age, and to identify major events that could have an effect on nitrogen fluxes. The project also made it possible to study root defects in autophagy mutants and establish a link between the root autophagy mechanism and water flux management in the plant.

Finally, the hAPPEN project identified protein partners that could participate in the autophagic machinery, which now needs to be studied.

The perspectives of the hAPPEN project are (i) the search for mechanisms regulating nitrogen fluxes at the whole plant level, (ii) the search for factors regulating autophagic activity that could serve as biostimulants of nutritional recycling, and (iii) the detailed dissection of the role of new potential autophagy partners identified in hAPPEN.

The hAPPEN project is a fundamental research project, coordinated by the Jean-Pierre Bourgin Institute for Plant Sciences (INRAE), in partnership with the EVA unit of the University of Caen Normandy. The project began in January 2020 and lasted 5 years. It received ANR funding of €463,265 for a total cost of €522,264.

Ammonia-nitrate fertilizers are responsible for the release of nitrous oxide and for water pollution. Reducing the use of nitrate fertilizers without affecting plant yields and product qualities could be achieved by increasing the nutrient-recycling performances in plant organs especially by improving N remobilization from senescing organs. Leaf senescence is a well-known developmental process important for the management of plant resources. During senescence, sequential degradation of cell components is chronologically tightly regulated and coordinated in order to recycle nutrients and proceed to remobilization to the sinks. Chloroplasts are the first organelles to be dismantled during leaf senescence and the major source of nitrogen in the proteins of the leaves.
Surprisingly, most of the senescence-associated proteases are located in lytic vacuoles. Among them we can cite the SAG12 (senescence-associated gene 12), RD21 and Cathepsin B cysteine proteases and the CND41-like AED1 aspartic protease. Except they are induced during leaf senescence, nothing is known about the role of these proteases in nitrogen recycling and remobilization, except in the case of SAG12 that was recently reported. The chronology of the expression and maturation of the different proteases and their localizations in the cell compartments are clearly strategic mechanisms to keep the catabolic processes and the onset and duration of leaf senescence strictly under control until cell death.
The degradation of the chloroplast stroma proteins has been studied for long. It consists of a fragmentation of stroma proteins after oxidation in the presence of reactive oxygen species. The fragmentation products likely persist in the chloroplast, until they are released through the stromule protrusion in vesicles, named RuBisCO-containing bodies (RCBs), and in senescence-associated vacuoles (SAVs) that contain the SAG12 cysteine protease. It was shown that the trafficking of RCBs to the central lytic vacuole requires the macro-autophagy pathway. Thus protease and autophagy cooperate in the proteolysis of chloroplast proteins.
The two groups from IJPB (SATURNE group) and UMR INRA-UniCaen EVA (INCCA group) involved in this project have shown that:
(i) Autophagy and the SAG12 cysteine protease can control nutrient mobilization for efficient seed filling in Arabidopsis;
(ii) Increasing autophagy in Arabidopsis can improve nutrient recycling and mobilization to sink tissues as seeds;
(iii) The well-known SAG12 leaf senescence-associated cysteine protease is unexpectedly strongly expressed in the roots of Arabidopsis, where it controls nitrogen fluxes from the root to seeds and thus sustains seed filling when plants face to N deficiency;
(iv) The role of SAG12 in the leaf during senescence is essential for leaf nitrogen mobilization and the expression of SAG12 in senescing leaves and also in roots at reproductive stage suggests it participates in shoot to root signaling, releasing leaf-senescence signals;
(v) Several proteins that accumulate in autophagy mutants specifically and that could be specific autophagy cargoes, as suggested by the presence of AIM motifs in their sequences and by their gene regulation networks.
Altogether results suggest that compensatory effects between the different leaf-senescence proteases) and the autophagy pathway facilitate efficient nutrient mobilization.
The objective of the project is to investigate the complementary roles of autophagy and plant proteases in the mobilization of nutrients, and in the sink/source signaling during leaf and root senescence.
This project integrates multiscale levels of studies, from cellular mechanisms to the whole plant level of resource allocation.