Sensl: a synthetic biology sensor for tracking strigolactone signaling in rice

Strigolactones (SLs), a special class of plant hormones and rhizosphere signals, play an indispensable role in regulating important agronomic traits of rice (Oryza sativa), one of the most important crops in the world (Wang et al., 2018; Chen et al., 2022). Genetically or chemically modifying the SL pathway could significantly alter plant architecture and crop yield of rice through the regulation of shoot branching (Zou et al., 2006; Arite et al., 2007; Lin et al., 2009; Wang et al., 2020). SLs can also coordinate with other plant hormones, such as auxin, brassinosteroid, gibberellin, and abscisic acid (ABA), to regulate rice growth and metabolic processes (Sang et al., 2014; Fang et al., 2020; Liu et al., 2020; Sun et al., 2023). Moreover, SLs have been shown to participate in integrating N, Pi, as well as sucrose signals to influence rice development and environmental adaptation (Shi et al., 2021; Patil et al., 2022; Barbier et al., 2023). In light of the pivotal role of SLs in rice breeding, it is necessary to develop precise molecular tools for real-time monitoring of SL signaling dynamics in rice. Up to now, strategies for in vivo and in situ tracking of SLs have been limited. Researchers largely depend on mass spectrometry (MS) to quantify SLs in plant cell lysate (Xie et al., 2013; Halouzka et al., 2020; Yoneyama et al., 2022), which is often compromised by SLs' limited stability, high MS costs, and scarce analytical standards (Floková et al., 2020). Recent advances in genetically encoded ratiometric reporter systems, such as StrigoQuant (Samodelov et al., 2016) and pRATIO (Khosla et al., 2020; White et al., 2022), along with engineered fluorescent biosensors utilizing SL receptors (Chesterfield et al., 2020), provide promising alternatives. However, the above-mentioned sensors are only successfully applied in transient expression systems, of which the application in intact plants has not been achieved. Although the fluorescent biosensor Strigo-D2 enables SL signaling monitoring in Arabidopsis seedlings (Song et al., 2022), there remains a critical gap in high-throughput, noninvasive tools for SL signaling detection in vital crops like rice, where SLs are essential for regulating several important agronomic traits. We first demonstrated that Sensl's response is dependent on bioactive SLs and proteasome activity. Next, we validated Sensl's ability to detect fluctuations in endogenous SL levels in intact living rice. We also pursued extensive Sensl exploration in distinguishing SL stereochemistry and detecting cross talk between SLs and other phytohormones. Additionally, we developed a streamlined in vitro method for assessing OsD53 degradation using Sensl. Collectively, our results highlight the significant value of Sensl as a versatile instrument for unraveling SL signaling in rice by combining species specificity, ratiometric precision, and in vivo–in vitro dual-mode compatibility. The study deployed rice (O. sativa L. ssp. japonica cv Nipponbare) as the wild-type (WT) background and for generating Sensl transgenic lines. Rice seeds were subjected to surface sterilization using 20% (v/v) sodium hypochlorite solution with 0.01% Triton X-100 for 20 min. Following sterilization, seeds were rinsed five times with sterile double-distilled water and then germinated for 30 h in sterile water at 28°C in the dark. Germinated seeds were hydroponically cultivated in Yoshida rice nutrient salt mixture (Coolaber, NSP1040) within a growth chamber maintained under controlled conditions (14 h : 10 h, 28 : 22°C, light : dark photoperiod, 200 μmol photons m−2 s−1). To assess Sensl's specificity and dose–time response to SLs, 5-d-old rice seedlings were sprayed with luciferase–hormone mixtures combining various SL analogs with 0.06 mg ml−1 d-Luciferin in a 1 : 1 ratio. To investigate the proteasome-dependent degradation of Sensl, 5-d-old rice seedlings were pretreated with 50 μM MG132 for 5 h before being sprayed with luciferase–hormone mixtures. For the Pi deprivation response assay 4-deoxyorobanchol (4DO) content detection, luminescence intensity analysis, and quantitative real-time polymerase chain reaction (qRT-PCR)), rice seedlings were initially cultured in a standard Pi nutrient solution for 12 d. Afterward, the seedlings were transferred to a standard Pi (NSP1040) or Pi-deficient (NSP1040-P) liquid medium and grown for an additional 2 wk (14 d) under identical temperature and light conditions in a growth chamber, replenishing the culture medium every 3 d. For assessing short-term hormonal responses, a range of plant hormones was mixed with 0.06 mg ml−1 d-Luciferin substrate in a 1 : 1 ratio. The solutions contained 50 μM ABA, 20 μM brassinolide (BL), 50 μM 1-aminocyclopropane-1-carboxylic acid (ACC), 10 μM GA3, 2 μM tZ, 10 μM jasmonoyl-isoleucine (JA-Ile), 2 μM indole acetic acid (IAA), 500 μM salicylic acid (SA), 1 μM rac-GR24, 2 μM KAR1, and 0.1% (v/v) dimethyl sulfoxide (DMSO). Five-day-old rice seedlings were sprayed with these mixtures. In the context of the long-term hormonal responses, 5-d-old rice seedlings were immersed in Yoshida rice nutrient solution supplemented with various hormones (at the same concentration of the short-term response) for 6 h. The specialized Sensl plasmid, based on the pUC19 backbone, was engineered and synthesized by the Beijing Genomics Institute. For optimal expression in rice plants, the DNA sequences encoding for firefly luciferase (FLUC) from Photinus pyralis and Renilla luciferase (RLUC) from Renilla reniformis were adapted using the O. sativa codon preference database accessible at http://www.kazusa.or.jp/codon/. Sensl encompasses a fusion of the OsD53 protein to FLUC, forming a sensor module. This is linked to the normalization reporter element, RLUC, through a self-cleaving 2A peptide. The entire construct is under the transcriptional control of a robust promoter derived from the rice ACTIN1 gene. For conducting bioluminescence imaging of Sensl responses, rice seedlings were sprayed with 0.3 mg ml−1 d-Luciferin, potassium salt (40902ES01; YEASEN, Shanghai, China). After a 5-min equilibration period, imaging was executed using the BLT PlantView100 (Guangzhou Biolight Biotechnology Co, Guangzhou, China). Quantitative analysis of the bioluminescence intensity was performed using ImageJ (2.1.0). The luminescence intensity within each region of interest (ROI) was meticulously quantified, ensuring ROI dimensions remained consistent across all images to facilitate accurate comparison. The YEASEN Dual-Luciferase Reporter Assay Kit (11402ES60) was employed for the examination of dual luciferase reporter genes. At the designated time points, tissue samples from rice seedlings under different conditions were collected and rapidly frozen in liquid nitrogen. Tissues were then pulverized using a homogenizer at 45 Hz for 30 s. Following the complete disruption, 300 μl of lysis buffer was added to the samples in Eppendorf tubes. The samples were then incubated on ice for c. 5 min to ensure thorough lysis of the material. The samples were centrifuged at 12 000 g (Thermo Fresco 17) for 1 min, and the supernatant was subsequently transferred. A 20 μl aliquot of the lysate was dispensed into a well of a white 96-well assay plate. First, 100 μl of firefly luciferase assay reagent was added, and the plate was shaken to mix. The activity of FLUC was measured. Following this, 100 μl of RLUC assay reagent was introduced, the plate was again shaken to mix, and the activity of RLUC was assessed. Assays were performed using the Spark multimode microplate reader (Tecan, Mannedorf, Switzerland). The cell-free protein degradation assay was conducted in accordance with the procedures outlined by Wang et al. (2009). Total proteins were isolated utilizing a degradation buffer comprising 25 mM Tris–HCl (pH 7.5), 10 mM NaCl, 10 mM MgCl2, 4 mM phenylmethylsulfonyl fluoride, 5 mM dithiothreitol, and 10 μM ATP. The proteasome inhibitor MG132 and various hormones were added to the mixture as specified. The samples were then incubated on a ThermoShaker TS1 with constant shaking (28°C, 300 rpm). At predetermined time points, a 30 μl aliquot of the reaction mixture was taken and instantly frozen in liquid nitrogen to halt the reaction. Subsequently, a dual-luciferase reporter assay kit was used to measure the luciferase activities. Plant samples were first ground in liquid nitrogen, and then, 100 mg of powder was accurately weighed and transferred into a 2-ml centrifuge tube. Extraction buffer of 1.5 ml (isopropanol: formic acid = 99.5: 0.5, v/v, with a final concentration of 2 ng L–1 of 13-13C-GR244DO as internal standard) was added with vortexing for resuspension of samples, and a 30 min ultrasonic treatment was applied to improve extraction efficiency. Then, after 15 min centrifugation at 14 000 g, 1.4 ml supernatants was transferred and dried in a LABCONCO CentriVap vacuum centrifugal concentrator and resuspended with 60 μl methanol solvent (water : methanol = 20 : 80, v/v). Then the contents of 4DO were detected by a triple quadrupole mass spectrometer and calculated with a calibration curve made with standards and internal standards. Positive ionization mode data was acquired using a Sciex Triple Quad™ 7500 LowMass quadrupole mass spectrometer (AB SCIEX) coupled with a Nexera Series (Shimadzu) UHPLC. Samples were separated with a 100 × 2.1 mm, 2.5 μm XSelect™ HSS T3 column (Waters, Milford, MA, USA). Five microliters of samples were loaded each time, and the flow rate was set at 300 μl min–1, with the column oven set at 40°C. Solvents for the mobile phase were 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B). The gradient of the mobile phase was: 0 min, 60% B; 0–1 min, 60%; 1–3 min, 40% B; 3–5 min, 20% B; 5–7 min, 0% B; 7–8.5 min, 0% B; 8.5–8.6 min, 60% B; 8.6–11 min, 60% B. The autosampler was set at 10°C. MS was operated in positive electrospray ionization mode. Multiple reaction monitoring mode was used for detection. The temperature of the Electrospray ion source was set at 350°C. Curtain gas flow was set at 40 psi, collisionally activated dissociation gas was set at 9, and the ion spray voltage was 3500 V, with ion gas 1 set at 30 psi and 2 set at 50 psi. Data acquisition and processing were performed with SclEX OS 3.1.6.44 software. Three-dimensional structures of GR245DS (PubChem CID: 11358436) and GR24ent5DS (PubChem CID: 10590166) were obtained from the PubChem database. The receptor structure of D14 (PDB ID: 5dj5) (Zhao et al., 2015) was downloaded from the Protein Data Bank (PDB). The spatial Data file (SDF) and PDB files underwent conversion to the PDBQT (PDB, Partial Charge (Q), & Atom Type (T)) format using the ‘prepare_ligand’ and ‘prepare_receptor’ utilities from the AutoDockFR suite (Morris et al., 2009). We used AutoGrid4.2.7 with default settings for grid parameter determination (Zhang et al., 2019). The grid dimensions were set at 20.7 × 13.0 × 17.0 Å, with a 0.375 Å spacing. Docking was executed at an of The were structures were using For of the was The expression data of OsD53 and other SL and under various were from the database and The expression obtained from the database were subjected to the rice seedlings grown hydroponically at 28°C under light were with plant hormones GA3, tZ, and acid seedlings hormones samples were collected at 0.5, and 6 h with was from samples, with for and for and using a expression were as of intensity and were using the 2022). The assay of expression with the extraction of by to the by with the of and as The quantitative analysis of expression was performed utilizing in with an real-time by a normalization the rice ACTIN1 was designated as the internal gene. The of ACTIN1 1 is to the and the is to the FLUC D14 and used for are in For the and analysis of the database was a of and a robust for such To between OsD53 and other we the of the to the We and our to the O. sativa species with a of and of the were using the processes within the were into were to proteins to The protein structure of RLUC and the protein structure of and in were all using The protein sequences of D14 and were from quantitative data were using with the and were employed to the data where the the range to with a the and the the data are as to the data a in rice tools to in SL signaling are limited. To the dynamics of SL signaling in a and precise in rice, we a protein SL signaling biosensor Sensl. a that degradation in response to SLs, was to FLUC as the sensor module. a used of in vivo the FLUC for noninvasive and dynamics of OsD53 RLUC was set as the normalization to with the sensor a 2A which can proteins the of of et al., as luminescence signals for FLUC and activity for The entire is as a under the of the rice promoter This could not only the SL signaling and FLUC bioluminescence imaging also quantitative analysis on SL signaling by luminescence of FLUC and RLUC dual luciferase reporter assay To the and of Sensl in rice, the of FLUC and RLUC were subjected to codon DNA sequences of FLUC and RLUC, from the pyralis and were for rice plants by the O. sativa codon database. To codon used were with used a the codon adaptation of the encoding FLUC and RLUC from and to and the and contents also to and from the and are to the content of O. sativa which is c. et al., This codon the of the Sensl sensor for application in rice. To the of ratiometric SL signaling sensor in rice, we transgenic Sensl in the background and with OsD53 expression as well as to of plants for To the of Sensl expression on endogenous SL we performed analysis to measure the expression levels of in SL and regulation in and wild-type were significant in the expression of these between the and wild-type plants that Sensl expression has on endogenous SL and in the of Sensl as a for monitoring SL signaling in Next, we the of Sensl 1 h. After the FLUC there was significant of the luminescence signals in the transgenic rice seedlings within 1 h the analysis of FLUC as by that the luminescence intensity remained within 30 min results that Sensl a detection time The biosensor for dynamics in and We first the quantitative between Sensl and SLs by monitoring the luminescence in the transgenic seedlings and of shown in 1 μM to luminescence of Sensl in rice imaging that the degradation was by bioactive rac-GR24, with the to in 30 min a to the of SLs, on the luminescence of Sensl Additionally, the of RLUC, as an internal was by the of significant in intensity between the control and treatment have shown that OsD53 is and the in response to et al., 2013; et al., we pretreated the transgenic rice seedlings with 50 μM MG132 for 30 min, which is used to the protein the degradation of was pretreated with MG132 reporter assay that with MG132 the of collected data that Sensl as a it is to the precise regulation of the protein degradation pathway within the Sensl is for monitoring the fluctuations of bioactive has been that the between Arabidopsis of and is (Wang et al., Sensl, we OsD53 to levels of the concentration of sprayed rac-GR24, the in signals After 30 min with 0.5, and 1 μM rac-GR24, the analysis of were c. and 20% of the control that Sensl is in a to SL Moreover, we the in signals the application of at different time analysis a in the luminescence signals within the min a significant was the min treatment which with that response to the et al., Collectively, these results that Sensl could the of SL signaling response in a which specificity for the detection of SL SLs are a class of plant hormones that plants with to from Pi or the endogenous of SLs plants to to nutrient et al., 2023). In we initially performed an MS analysis to measure the rice SL 4-deoxyorobanchol (4DO) content in Sensl transgenic plants under Pi-deficient the Pi-deficient the 4DO was detected at 60 However, the 4DO content under conditions our standard detection and it is to 6 To the of Sensl to in endogenous SL signals, we then Sensl's SL signaling in response to Pi-deficient The luminescence under Pi-deficient conditions were under conditions Moreover, the Pi-deficient treatment was by c. are consistent with results by molecular that the expression of a set of in SL is by Pi et al., et al., 2023). To these we performed analysis to detect the of SL and and in Sensl transgenic and were used as positive (Xie et al., 2022; et al., 2023). shown in the expression of and was significantly under Pi we that the of Sensl to endogenous SL Up to now, SL with between the and have been in the plant et al., 2023). the of a SL not SL signaling in analysis of with D14 that the of GR245DS with the and a to D14 at GR24ent5DS to D14 data are consistent with that the receptor D14 to SL not To Sensl can to the of SL signaling in plants, we the transgenic seedlings with GR245DS and GR245DS significantly the luminescence intensity of within 30 min, which to that of GR24ent5DS a on the luminescence of the luminescence the of Sensl on GR245DS The of Sensl signals that the a SL signaling Sensl, with high in could as a for distinguishing the of SLs and SL In we detected Sensl response in the of an SL from the linked et al., 2016) and that the could not the degradation of and between are vital for plant growth and has been that SLs other plant hormones to regulate plant development and et al., 2022). However, these hormones or SL signaling remains data from the we the expression of OsD53 with various plant hormones μM 10 μM acid 3 50 μM ABA, 1 μM 1 μM and 100 μM at different time hormones not in the levels of OsD53 within 1 h. However, with the expression of OsD53 that regulation in the expression of OsD53 the of SL we that a long-term treatment influence SL To the of other on the SL we the response of Sensl to various plant hormones in First, rice seedlings were with different bioactive μM μM GA3, 2 μM 50 μM ABA, 20 μM 50 μM 10 μM 500 μM 2 μM as well as 1 μM a to OsD53 expression results only could a response in Sensl. In the biosensor not to other plant hormones We the of the SL in response to a A in luminescence intensity was with 50 μM for 6 h, in with the from the OsD53 expression to 10 μM or 10 μM in in Sensl signals To into the by which SL we the expression data of from the database. h we a in the expression of in SL 20 and and in rice as by the data to the at 0 h the expression of which a in and SL significantly in with a regulation of as in et al., In the SL receptor D14 remained to the in Arabidopsis et al., that SL signaling by The specificity of plant regulation the of Sensl as a of a for the between SLs and other In to detecting SL signaling in we also an in vitro cell-free protein degradation assay for OsD53 by Sensl or proteasome inhibitor were added to protein from Sensl incubated for 3 h, and subjected to dual luciferase reporter assay at different time with data from in vivo degradation of OsD53 as was by treatment with the proteasome inhibitor MG132 as a of in vivo the application of not significantly the of which is to that treatment data a in which SLs degradation in the of Sensl signaling by in vivo through an on the between and SL utilizing Sensl for in vitro cell-free degradation we could influence SL signaling a method that and in The development of Sensl, the first ratiometric sensor for real-time monitoring of SL signaling dynamics in rice, a significant in the of plant SL signaling which are largely limited to transient expression (Samodelov et al., et al., 2020), Sensl enables tracking of SL signaling dynamics in intact rice plants within a critical for crop with which to SL signaling in Arabidopsis (Song et al., 2022), Sensl FLUC a in plant where often with detection. Moreover, Sensl's ratiometric FLUC and RLUC, the and of SL it a versatile for in vivo and in vitro Sensl is not an Sensl on degradation of the OsD53 protein to SL signaling detecting the SL high and specificity, it also as the expression of OsD53 with endogenous SL We can the of different plant as well as on SL signaling by between and wild-type Sensl or by analysis of the same before and after Sensl significant for SL Moreover, the of Sensl is to the of protein it has not been validated through real-time monitoring could these by OsD53 with or biosensors that of the SL signaling from and could the of such and from combining Sensl with such as or of could for the of SL For our analysis of the to OsD53 in processes and molecular with OsD53 a for into the molecular SL the of such as and protein could the of biosensors with and with signaling et al., these in plant biosensor yield and This was by the of the of and and was also by the of the of and the of This has been by the through the and the and the performed most of the and in and the and the final The data study are in the expression data of OsD53 and other under from database and and quantitative real-time polymerase chain reaction for and of FLUC and of transgenic rice Sensl. levels of SL in and wild-type time of Sensl and of FLUC and The expression of SL is by Sensl the of different of different plant in rice. In vitro protein degradation assay by Sensl. and of OsD53 expression data from database. data of from database. of used in monitoring of Sensl response to is not for the content or of by the to the The is not for the content or of by the to the for the The remains with to in and in