Investigations of plant salt stress responses using genetically-encoded fluorescent reporters
Final Report Abstract
The basic module aimed to fund the further development of ABAleon2.15, a FRET-based fluorescent indicator for the plant hormone abscisic acid (ABA). In addition, other types of multispectral ABA indicators were supposed to be developed based on the prototype ABAgreen-mCherry. The newly developed ABA indicators were then supposed to be transformed into Arabidopsis thaliana in order to facilitate in planta analyses of ABA. Within this project, I have designed, engineered and tested several ABA indicator candidates after transient expression in tobacco (Nicotiana benthamiana), human embryonic kidney HEK293T cells and E. coli, and after stable expression in Arabidopsis thaliana plants. From the various approaches that I have pursued, I was not successful with the establishment and development of ABAgreen-mCherry variants. However, for the development of FRET-based ABA indicators, several approaches were pursued, including the testing of various fluorescent protein FRET pairs, ABA-binding sensory domains, linkers within the sensory domain and linkers from the sensory domain to the fluorescent proteins. This comprehensive approach, combined with the establishment of a testing platform in HEK293T cells resulted in the development of the ABA indicator ABAleonSD1-3L21. Compared to the previous version ABAleon2.15, this indicator exhibited increased ABA-induced emission ratio changes in HEK293T cells. However, the results could not be supported by in vitro analyses. Yet, in vivo analyses in stably expressing Arabidopsis plants indicated that ABAleonSD1-3L21 responded in ABA uptake experiments slightly faster to ABA and with slightly larger emission ratio changes compared to ABAleon2.15. Although the ABA response parameters in Arabidopsis did not seem to be significantly improved, I still recommend using ABAleonSD1-3L21 in future experiments. Therefore, I have already established Arabidopsis dual-sensing reporter lines that simultaneously express ABAleonSD1-3L21 together with red fluorescing indicators for pH, Ca2+ and H2O2 (RW341-RW343 and RW355-RW357 can be found in the appendix). In the future, I aim to use ABAleonSD1-3L21 to investigate the spatiotemporal parameters of ABA synthesis and ABA fluxes in response to environmental changes, such as drought, heat, cold, flooding, salt- and osmotic stress. In addition, I aim to determine the function of ABA transporters in these processes. In combination with other red fluorescing indicators, I aim to use ABAleonSD1-3L21 to perform research on how ABA signals are coordinated with and dependent on other signaling processes, such as pH, Ca2+ and H2O2 signals.
Publications
- 2017. Multiparameter imaging of calcium and abscisic acid and high-resolution quantitative calcium measurements using R-GECO1-mTurquoise in Arabidopsis. New Phytol 216(1): 303-320
Waadt R, Krebs M, Kudla J, Schumacher K
(See online at https://doi.org/10.1111/nph.14706) - 2017. SnapShot: Abscisic Acid Signaling. Cell 171(7): 1708-1708.e1700
Hauser F, Li Z, Waadt R, Schroeder JI
(See online at https://doi.org/10.1016/j.cell.2017.11.045) - 2018. Abscisic acid-independent stomatal CO2 signal transduction pathway and convergence of CO2 and ABA signaling downstream of OST1 kinase. Proc Natl Acad Sci USA 115(42): E9971-E9980
Hsu PK, Takahashi Y, Munemasa S, Merilo E, Laanemets K, Waadt R, Pater D, Kollist H, Schroeder JI
(See online at https://doi.org/10.1073/pnas.1809204115) - 2018. Genetically Encoded Biosensors in Plants: Pathways to Discovery. Annu Rev Plant Biol 69: 497-524
Walia A, Waadt R, Jones AM
(See online at https://doi.org/10.1146/annurev-arplant-042817-040104)
