Development And Application Of Organellar-Specific ABA Biosensors

As sessile organisms,plants have evolved to cope with numerous environmental challenges such as soil salinity,drought and extreme temperatures through timely and effective regulation of metabolic processes for fine-tuning their growth.As one of the key phytohormones,ABA plays essential roles in plant stress responses.Many environmental stresses(e.g.drought,salt damage,etc.)trigger a rapid and significant accumulation of ABA in plant cells.Cellular ABA accumulation activates various responses in a diverse array of cell types,such as promoting stomatal closure and inhibiting seed dormancy.When the stressful environment is relieved,intracellular ABA rapidly returns to its basal levels.Thus,the regular changes in plant physiological and environmental conditions keep intracellular ABA levels in a constant dynamic equilibrium.Plant cellular ABA levels are regulated through complicated mechanisms,including ABA biosynthesis,catabolism,reversible glycosylation,and intercellular and intracellular transmembrane transport between different organelles.And this complex ABA regulation involves different subcellular organelles as well as adjustments at different levels(regulation of transcription,translation,post-translational modifications,and enzymatic activities of various proteins involved in the above processes).Considering the limited subcellular location of its receptors,it is speculated that ABA-mediated signaling is largely dependent on local ABA levels rather than the overall ABA concentration within a plant cell.Thus,monitoring changes in ABA levels in different organelles in a timely and sensitive manner is essential to elucidate ABA-mediated signal transduction.Despite that the fluorescence resonance energy transfer(FRET)-based ABA biosensors have been developed and are able to detect intracellular ABA concentrations,direct imaging of subcellular ABA levels remains unresolved.In this study,we modified the reported ABA biosensor ABAleon and targeted it to different organelles for monitoring organellar ABA levels under different environmental conditions.The main findings are as follows:1.The reported ABAleon2.1 was modified based on nanobody-epitope interactions and a new ABA biosensor,named ABAleon2.1_Tao3(abbreviated as Tao3),was generated and contains the epitope sequence SYN.Meanwhile,the endoplasmic reticulum(ER)membrane anchoring protein with the NbS was generated.When co-expressed together with the ER membrane anchor in a transient expression system in tobacco protoplasts,ABAleon2.1_Tao3 was targeted to the ER membrane through nanobody epitope-mediated protein interactions,wherein the ABA biosensor units were faced to the cytoplasm(aTao3)and ER lumen(aTao3ER),respectively.2.ABAleon2.1_Tao3 was also targeted as soluble chimeras to different subcellular organelles,including ER,chloroplast/plastid and nucleus,through traditional organellar targeting,namely,through addition of organellar-specific signal peptides to the N-terminal of ABAleon2.1_Tao3.The correct targeting of these soluble organellar ABA sensors were verified both in tobacco mesophyll protoplasts as well as in transgenic Arabidopsis plants.3.Combined FRET with fluorescence lifetime imaging microscopy(FLIM),the changes in the fluorescence lifetime,an intrinsic feature of fluorescence proteins,of the donor fluorescent protein mTurquoise(mT)in ABAleon2.1_Tao3s induced upon ABA treatment were detected and calculated in relative to alterations in the level of organellar ABA.In tobacco protoplasts from mesophyll cells,FRET-FLIM analysis of the ER membrane anchored Tao3s revealed that the basal level of ABA in the ER lumen was generally higher than that in the cytoplasm,which was somewhat unexpected.More importantly,upon different stress treatments,the ER ABA responded distinctly to different stimuli,whereas the ABA level in the cytosol was generally enhanced.4.In the roots of aTao3 and aTao3ER stably transformed Arabidopsis plants,mannitol treatment triggered an increase in the level of cytosolic ABA in meristematic zone cells and of the ER ABA in the elongation/maturation zone cells within 1 h after treatment.In comparison,ABA levels at both places no longer changed in the bg1-2 mutant,indicating that osmotic stress-induced ABA accumulation in those two cell types in Arabidopsis roots is dependent on BG1.5.Taking advantage of soluble organellar Tao3s,different subcellular ABA distribution patterns were revealed in different tobacco cells.Besides,in tobacco mesophyll cells and Arabidopsis cells from distinct tissues,organellar ABA showed stimulus-dependent changes upon stresses.For instance,the Arabidopsis cells from both meristem and elongation cells accumulated notable ABA in the cytoplasm,whereas the plastid and nuclear ABA was generally decreased after mannitol treatment.In Arabidopsis leaf tissues,organellar ABA appeared to be universally hyposensitive to stress treatments,with a significant increase in the ER ABA in epidermal cells and a decrease in the nuclear ABA in guard cells.6.Through screening,an ABCG(ATP-binding cassette transporter family,G group)mutant abcg16 was found to be distinctly different in endogenous ABA level at normal and stressed conditions compared with wild-type Col-0,implying that ABCG 16 played a positive and a negative role in ABA signaling under normal growth and osmotic stress conditions,respectively.Utilizing organellar ABA sensors and tobacco protoplast system,it was found that ABCG16,like its homologues ABCG25,functioned as an ABA exporter mediating cellular ABA efflux.Meanwhile,based on the nuclear envelop(NE)/ER membrane localization of ABCG16,it was further revealed that ABCG 16 facilitated the entry of ABAGE,instead of ABA,into the ER lumen,thereby enhancing ABA levels in the ER and the nucleus.These results support that ABCG 16 could also be a transporter of ABA-GE for promoting the entry of ABA-GE in the ER,thus mediating ABA equilibrium between cytoplasm and the ER/nucleus.In summary,our study utilized two different organellar targeting strategies,one of which is particularly integrated with the nanobody technology,and developed membrane anchored and soluble ABA sensors targeted to different subcellular organelles.With these organellar ABA sensors,we revealed not only distinct organellar ABA distribution patterns in different plant cells,but also stimulus-dependent changes in organellar ABA upon environmental stresses.Moreover,our study established the ABCG protein ABCG 16 as a dual transporter for both ABA and ABA-GE,thus mediating ABA signaling under normal and stressed conditions.It lays the foundation for elucidating the molecular mechanisms of ABA-mediated signal transduction and regulation of different physiological processes under drought stress conditions,provides a theoretical basis for improving plant drought resistance,and provides practical guidance for molecular breeding for crop stress resistance.