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

Abstract

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 m

Plant phenotyping relevance

イネ体内のストリゴラクトンシグナルを非侵襲・定量的に追跡する生物発光センサーを開発し、実植物での検出性能を検証しており、フェノタイピング手法が研究の中心である。

Code and data availability

The paper reports a rice strigolactone biosensor (SenSL) with luminescence imaging, qRT-PCR, docking, and PPI analyses. No authors' public code, datasets, images, or trained models are deposited; the only data availability statement points to supporting information tables (RiceXPro-derived expression data and qPCR prim

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