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Growth balance regulation by SnRK1 under ABA-stress conditions

Periodic Reporting for period 1 - ABA-GrowthBalance (Growth balance regulation by SnRK1 under ABA-stress conditions)

Reporting period: 2019-10-01 to 2021-09-30

ABA-related abiotic stresses such as drought, salinity and extreme temperatures, pose serious limitations to plant growth, reducing crop productivity between 50% and 80%. This is aggravated by the necessity to increase global crop yields by 70% to meet the demands of a constantly growing world population (9 billion people in 2050). Given that drought is the main environmental factor affecting yields, the development of plant varieties with greater water use efficiency and drought tolerance could significantly increase agricultural production. However, despite our increasing knowledge on individual stress signalling pathways, how they interact with each other and how they modify growth is still poorly understood. The central hypothesis of the ABA-GrowthBalance project is that the interplay between energy and ABA signalling is important for the successful orchestration of ABA responses and growth repression in stress. This project is therefore expected to provide molecular understanding on the trade-offs between stress tolerance and growth/yield, potentially contributing to more successful crop improvement where these two processes can be uncoupled and engineered in a more targeted manner.

The main aim of this project was to uncover the mechanisms by which Snf1-related protein kinase 1 (SnRK1) affects ABA signalling and to investigate the relevance of the ABA-SnRK1 crosstalk for controlling the activity of Target of Rapamycin (TOR) and/or other growth regulators in Arabidopsis thaliana.

This project provided novel molecular insight on the trade-offs between stress tolerance and growth conclusing with the following model of regulation of the root growth by SnRK2 and SnRK1 kinases in favorable and stress conditions (see attached image): under optimal conditions, SnRK1α1 is sequestered in nuclear SnRK2-containing repressor complexes that harbor also a clade A PP2C. Sequestration of SnRK1α1 in these complexes is important to prevent SnRK1α1 activity and its exit from the nucleus. This enables TOR activity in the cytoplasm, allowing cell proliferation of the root meristem and root growth. Accumulation of ABA under stress conditions leads to disassembly of SnRK2 and PP2C-containing SnRK1α1 repressor complexes through canonical ABA signaling involving the sequestration of PP2Cs by the ABA-bound PYR/PYL/RCAR receptors. Disassembly of the complexes releases SnRK2 and SnRK1α1 with the latter being subsequently exported to the cytoplasm to inhibit TOR and downregulate cell proliferation and growth. TOR inhibition may also involve co-participation of SnRK2 kinases released from PP2C inhibition in the cytoplasm
Summary of the results:

- We determined that the impact of SnRK1 on ABA signalling is specific to certain aspects: early post-germination growth and root development.
- We identified the tissues where SnRK1-SnRK2 are localized and the ABA-effects on the cellular/sub cellular localization of SnRK1-SnRK2: in response to ABA, activated SnRK1 relocalizes from the nucleus to the cytoplasm meanwhile ABA treatment caused a significant reduction in the overall levels of SnRK2.2 in the root apical meristem while not affecting to its normal subcellular localization pattern.
- We established the in planta SnRK1-SnRK2 interaction and its dependency on ABA signaling: SnRK1-SnRK2 interaction decreases upon ABA application; SnRK1-SnRK2 interaction in the nucleus strictly depends on the presence of the PP2Cs.
- We discovered that although SnRK2s promote SnRK1 signaling, this does not appear to involve direct SnRK1α1 activation.
- We partly elucidated the SnRK1 ABA-interactome.
- We determined that SnRK1 and SnRK2s play a role in TOR repression in response to ABA: SnRK1 is required for direct ABA-induced TOR repression meanwhile SnRK2s seems to be only required for repressing TOR via SnRK1 in response to ABA.

Dissemination activities:

- Scientific papers:

1. Belda-Palazon B (corresponding autor), Costa, M, Beeckman, T, Rolland, F, Baena-González, E. ABA represses TOR and root meristem activity through nuclear exit of the SnRK1 kinase. bioRxiv (2021) https://doi.org/10.1101/2021.12.27.474243.
2. Belda-Palazon B, Adamo M, Valerio C, Ferreira L, Confraria A, Reis-Barata D, Rodrigues A, Meyer C, Rodriguez PL, Baena-González E. A dual function of SnRK2 kinases in the regulation of SnRK1 and plant growth. Nature Plants. 2020 October 19. https://doi.org/10.1038/s41477-020-00778-w

- Oral communications:

1. Selected talk at Society of Experimental Biology (SEB) 2021 Annual Conference. Title: ABA promotes nucleus-to-cytoplasm relocalisation of SnRK1 to inhibit TOR. Virtual (07/06/2021).
2. Invited conference at Max Planck Institute. Title: Growth balance regulation by SnRK1 under ABA-stressed conditions. Potsdam, Germany (20/01/2021).
3. Invited conference at 2nd Young Researcher Symposium of the IBMCP. Title: Growth balance regulation by SnRK1 under ABA-stressed conditions. Valencia (11/11/2019).

Posters:

1. Belda-Palazon B, Costa M, Rolland F, Baena-Gonzalez E. ABA promotes nucleus-to-cytoplasm relocalisation of SnRK1 to inhibit TOR. EMBO Workshop Target of rapamycin (TOR) signaling in photosynthetic organisms. Virtual (21/10/2021-24/10/2021).
The study resulted from the ABA-GrowthBalance uncovered the mechanisms by which this happens: ABA signals are linked to a highly conserved regulatory system constituted by two protein kinases (SnRK1 and TOR), that control growth in all eukaryotes (animals, plants, fungi, and protists). When conditions are favorable the accelerator of the system (TOR) is active, inducing biosynthetic processes, cell proliferation and growth. When conditions are unfavorable the break of the system (SnRK1) becomes active, inhibiting TOR and consequently growth. This ancient system is controlled in all eukaryotes by nutrient signals, resulting in growth arrest when nutrient levels (“fuel”) are low. We found that in plants this system is controlled by additional signals related to the water status (ABA), conferring plants the unique capacity to regulate growth not only in response to nutrient signals but also in response to water availability. This system may have been crucial for the establishment of terrestrial life by maintaining resource spending and growth to a minimum when water was scarce. Importantly, our results unveiling the molecular connection between SnRK2, SnRK1 and TOR represents a significant advancement to the field of plant growth regulation and to the establishment of growth-defense trade-offs: (i) it brings mechanistic insight into the poorly understood effect of ABA on cell proliferation and root growth; (ii) it provides novel insight on how the SnRK1 kinase is regulated, underscoring the importance of its physical sequestration in specific subcellular compartments (in this case sequestration by SnRK2 kinases in the nucleus); (iii) It defies the established dogma that activation of SnRK1 leads to its translocation to the nucleus. Using the model plant Arabidopsis thaliana, we observed that when the SnRK1 kinase is genetically inactivated, plants develop larger roots under suboptimal conditions. Although such uncontrolled growth may be fatal under severe drought, it is likely to increase the capacity to absorb water from the more superficial soil layers, potentially improving plant growth when water is moderately limited.
Regulation of root growth by SnRK2 and SnRK1 kinases in favorable and stress conditions