Arabidopsis thaliana (Col-0) was grown in a growth chamber (LPH-240SP, Nippon medical & chemical instrument Co., Ltd., Osaka, Japan) for 4 to 6 weeks at 20 to 22 ºC and 70% humidity under a 12-h-light/12-h-dark cycle (66 µmol m− 2 s− 1 PAR).
Seeds of Samanea saman were purchased from World Flower Service Co., Ltd. S. saman trees were grown in a growth chamber (LPH-1PH, Nippon medical & chemical instrument Co., Ltd. Osaka, Japan) under a 16/8-h light/dark regime at an intensity of 160 to 290 µmol m− 2 s− 1 photosynthetically active radiation (PAR), at 25 ºC ± 3 ºC, and 70% relative humidity (the details of light and temperature setting for the growth chamber are: ZT 0, 20% intensity of light, 25 ºC; ZT 1:00, 50% intensity of light, 27 ºC; ZT 5, 100% intensity of light, 27 ºC; ZT 11, 50% intensity of light, 27 ºC; ZT 15, 20% intensity of light, 25 ºC; ZT 16, dark, 22 ºC). The study on this plant species has comply with relevant institutional, national, and international guidelines and legislation.
Preparation of Samanea motor cell protoplasts for ROS detection
Tertiary pulvini protoplasts were isolated from third to fifth branch from the shoot apex of the Samanea trees during ZT 3–5 according to the previously reported method 26,56,57, with modifications. About 100 of the tertiary pulvini were separated into extensor (adaxial) part and flexor (abaxial) part with a sharp razor blade and placed in 1 mL of predigestion solution (Gamborg’s B-5, 0.3 M sorbitol, 50 mM MES-KOH (pH 5.5), 0.2% BSA, 8 mM CaCl2). The osmotic pressure of the predigestion solution was then raised to 0.6 M sorbitol in two steps over 20 min with osmotic adjustment solution (Gamborg’s B-5, 4.0 M sorbitol, 50 mM MES-KOH (pH 5.5), 0.2% BSA, 8 mM CaCl2). Tissues were then moved into a ϕ 35 mm tissue culture dish with 1.6 mL filtered enzyme solution (Gamborg’s B-5, 50 mM MES-KOH (pH 5.5), 0.4 M sorbitol, 0.2% BSA, 8 mM CaCl2, 3% (w/v) each of Driselase (Aska Pharmaceutical Co. Ltd., Tokyo, Japan), Macerozyme R-10 and cellulase Onozuka RS, 0.3% pectolyase Y-23 (Yakult Pharmaceutical Industry Co., Ltd., Tokyo, Japan). Tissues in the enzyme solution were incubated with mild shaking for 1 h at 30°C, then without shaking for 1 h at 30°C. The enzyme solution was discarded and the tissues rinsed with recovering solution (3 x 1 mL, Gamborg’s B-5, 0.35 M sorbitol, 20 mM MES-Tris (pH 5.5), 100 mM KCl, and 1 mM CaCl2). The protoplasts were released in 1.6 mL recovering solution for 0.5-1 h at 30°C and debris removed by filtration of the protoplast suspension through a 50 µm nylon mesh. This step was repeated twice. The collected protoplasts were incubated at room temperature (24 ºC) for 3–4 h. Afterward, the protoplasts were concentrated on a sucrose cushion (0.57 M sucrose, 20 mM MES-Tris (pH 5.5), 10 mM KCl, 1 mM CaCl2) by centrifugation at 60 × g for 5 min and subsequently further purified on sucrose gradient: protoplasts were suspended with 0.8 mL ~ 80% sucrose cushion in a 2 mL Eppendorf tube, then 0.5 mL mix solution (sucrose cushion : wash solution = 4 : 3, wash solution: 0.57 M sorbitol, 20 mM MES-Tris (pH 5.5), 10 mM KCl, 1 mM CaCl2) was layered on top of the protoplast suspension and 0.5 mL wash solution was layered in upper part. The gradient was centrifuged at 130 × g for 10 min. The purified protoplasts were collected at the interphase between wash solution and mixed solutions. The yield of protoplasts was 1.5 to 3 × 105.
Measurement of ROS for motor cell protoplasts of S. saman using CLSM
The protoplasts were isolated as described above. Prepared protoplasts (10,000 cells/mL) in 130 µL wash solution were sealed in a glass-bottom Petri dish (φ 35 mm × 12 mm) coated with 200 µL of H2O and incubated overnight at 24 ± 1 ºC in dark. Then, the protoplasts were added to 5 µM H2DCF-DA (Sigma-Aldrich Co., Ltd., MO, USA), incubated for 45 min to stabilize their initial fluorescence intensity, and imaged by CLSM (LSM 700, Carl Zeiss, Oberkochen, Germany) at 2-min intervals for 20 min after treatment with 100 µM JAG 26 dissolved in 0.1% DMSO, H2O2 (FUJIFILM Wako Pure Chemical Industries Co., Osaka, Japan) at the indicated concentration, mock (0.01% ethanol or DMSO for DPI), or blank (untreated with any chemicals). When used, 12.5 µM DPI (Sigma-Aldrich Co., Ltd., MO, USA) dissolved in DMSO or 100 U/ml catalase (Sigma Co., Ltd.) was added 30 min before treatment with the above compounds. Intercellular fluorescence was excited using 488 nm light emitted by a solid-state diode laser at 0.5% with a Plan-Apochromat 40×/1.3 oil immersion objective and other settings as follows: emission 495–628 nm, master gain 500–650, pinhole 0.9 µm, 8-bit, frame 1024 × 1024 pixel, zoom 1.0, pixel dwell time 1.58 µsec/pixel and line average of 4. Autofluorescence was negligible in this emission range using these settings. Zen 2012 Black Edition software (Carl Zeiss, Oberkochen, Germany) was used for image analysis. Photobleaching and dye leakage from the intercellular to adjacent areas was too low to detect under these conditions. Dye leakage from the cytosol to the vacuole was assessed by comparing the levels of vacuole fluorescence at the end and beginning of each experiment. The round protoplasts were selected in which vacuole fluorescence intensity was less than twice as strong as that of background solution. However, the protoplast was discarded if dye leakage from the cytosol to the vacuole had increased the intensity of vacuole fluorescence up to 200%. ROS accumulation was calculated based on the fluorescence intensity of H2DCF-DA. The ROS accumulation of H2DCF-loaded protoplasts induced by blue light and the dark conditions was recorded with none-treatment as blank. ROS accumulation was estimated with the following equation:
(Fcn = the fluorescence intensity of a protoplast treated with chemicals at nth minutes. n = 0, 2, 4, … 20. Fuave.of n = average of the fluorescence intensity of untreated protoplasts at nth minutes. Fc0 = the fluorescence intensity of a protoplast treated with chemicals at 0 minutes. Fuave.of 0 = average of the fluorescence intensity of untreated protoplasts at 0 minutes). Pixel intensities of fluorescence at each given time were collected as the average intensity of three points that were away from the chloroplasts and vacuoles in each cell. Data were collected from two experiments in parallel on the same day.
Measurement of cell shrinkage for motor cell protoplasts of S. saman using CLSM
The protoplasts selected for measuring the cytosol fluorescence intensity were used for measuring the cell shrinkage. First, the intensity of the ROS signal was adjusted into similar-level contrast in the same protoplasts at the denoted times. Then, a red circle was made to fit the edge (critical surface of ROS signal) of the round part of the protoplast. The area of the red circle was calculated based on the average area of two independent fitting processes, and the changes in normalized protoplast volume calculated.
Quantitative RT-PCR analysis of SPORK2
To analyze time-course gene expression profiles of SPORK2 in Samanea tertiary pulvini, excised extensor and flexor motor cells were sampled every 2 hours from ZT 8 to ZT 14. Total RNA was isolated using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany), and cDNA was synthesized using ReverTra Ace (TOYOBO, Osaka, Japan) with oligo(dT)20 primers. Quantitative PCR was performed on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, CA, USA) with KAPA SYBR Fast qPCR Kit (KAPA Biosystems, MA, USA). The following primers were used; forward, 5’-TGCTGGTAAAATCACCAATACC-3’, reverse, 5’- GCCGTGATAAATTATCACAC-3’.