The latest HyPe(r) in plant H2O2 biosensing

HyPer7 senses minute amounts of H2O2 independent of pH and the glutathione redox potential and enables detection of physiological H2O2 fluxes within the cytosol and between subcellular compartments.

. Effect of GSH depletion on the oxidation of genetically encoded H 2 O 2 probes. A, Phenotype of Arabidopsis seedlings stably expressing HyPer7, HyPer or roGFP2-Orp1 five days after germination on MS-plates (Control; -) or MS-plates supplemented with 1 mM L-Buthionine sulfoximine (BSO; +) as an inhibitor of glutathione biosynthesis. B, False color ratio images of leaves, hypocotyl (Hyp), and root tips from the seedlings shown in (A). Ratio images were calculated from two fluorescence channels (Ch1: λ ex = 488 nm; λ em = 508-535 nm and Ch2: λ ex = 405 nm; λ em = 508-535 nm). Ratios for HyPer and HyPer7 are calculated as 488 nm / 405 nm, and 405 nm / 488 nm for roGFP2-Orp1. C, Ratio values from leaf, root and root tip samples. Box = interquartile range between the lower and upper quartiles, center line = median, + = mean, whiskers = min and max values. n = 3-9. Dashed lines indicate ratios of fully oxidized sensors after treatment with 10 mM H 2 O 2 , or fully reduced sensors after incubation with 10 mM DTT, respectively. Because HyPer7 cannot be reduced by DTT, the lower dashed line indicates the minimum ratio measured at steady state in non-stressed seedlings.  Supplemental Figure S4. Determination of minimum and maximum oxidation of different H 2 O 2 probes. A, Ratio images of different tissues from 7-day-old seedlings stably expressing HyPer7, HyPer or roGFP2-Orp1 sensors in the cytosol. The false color ratio images were calculated from two fluorescence images (Ch1: λ ex = 488 nm; λ em = 508-535 nm and Ch2: λ ex = 405 nm; λ em = 508-535 nm). Ratios for HyPer and HyPer7 are calculated as 488 nm / 405 nm, and 405 nm / 488 nm for roGFP2-Orp1. Seedlings were vacuum infiltrated with either imaging buffer (Control) or buffer supplemented with 10 mM DTT or 10 mM H 2 O 2 to fully reduce or oxidize the sensors, respectively. B-C, Ratio values from the leaf (B) and root elongation zone (C). Box = interquartile range between the lower and upper quartiles, center line = median, + = mean, whiskers = min and max values. σ = dynamic range of each sensor, calculated from the ratios values between fully reduced and fully oxidized probes. n = 6-11.  Figure S5. Hydrogen peroxide causes acidification in the cytosol. A, Ratio images calculated from confocal images of leaf, root elongation zone and root tip of 7day-old Arabidopsis seedlings expressing cpYFP in the cytosol. The false color 488 nm/405 nm ratio images were calculated from two individual fluorescence images (Ch1: λ ex = 488 nm; λ em = 508-535 nm and Ch2: λ ex = 405 nm; λ em = 508-535 nm). Seedlings were vacuum infiltrated with 10 mM H 2 O 2 or buffer as control. B, Quantitative analysis of ratio values. Box = interquartile range between the lower and upper quartiles, center line = median, + = mean, whiskers = min and max values. n = 3-8. Arabidopsis seedlings with stable expression of the HyPer variants were grown on agar plates for five days and incubated for 1.5 hours in solutions containing 50 mM NaAc and buffers with different pH. The cytosolic pH was estimated by using plants expressing the pH sensor pHluorin (see Supplemental Methods). Fluorescence was recorded by confocal microscopy (Ch1: λex = 488nm; λem = 505-550 nm; Ch2: λex = 405 nm; λem = 505-550 nm). Data show mean ratio values obtained from pH-clamped root cells and error bars correspond to the 95% confidence interval. n = 10-12 cells from two biological replicates. After around 1 h, MV was added to a final concentration of 50 µM (indicated by the arrow). Sensor oxidation was recorded as the normalized ratio of the fluorescence in two channels (Ch1: λ ex = 400±5 nm; λ em = 520±5 nm and Ch2: λ ex = 482±8 nm; λ em = 520±5 nm). Ratios for HyPer and HyPer7 are calculated as 482 nm / 400 nm, and for roGFP2-Orp1 as 400 nm / 482 nm. After a pre-incubation with MV for 2 h, seedlings were intermittently illuminated for 1 h with actinic light (200 µmol m -2 s -1 ) and measurements were subsequently resumed for additional 2 hours. Data represent mean ratios + SD, n = 3-4. B, Leaf disks from six-week-old plants stably expressing the indicated sensors were placed in a 96-well plate in imaging buffer. The arrow indicates addition of flg22 to a final concentration of 10 µM. Sensor oxidation was recorded as in (A). Data represent mean ratios + SD, n = 4-8. All ratio values were normalized to the ratio at t = 0 min.

Plant material and growth conditions
Arabidopsis (Arabidopsis thaliana) Col-0 ([L.] Heynh.) seeds were obtained from NASC (www.arabidopsis.info). Transgenic Col-0 lines expressing HyPer and roGFP2-Orp1 targeted to the cytosol has been described earlier by (Costa et al., 2010;Nietzel et al., 2019). For experiments with seedlings, seeds were surface-sterilized with 70% (v/v) ethanol, rinsed three times with sterile deionized water and stratified for 48 h at 4ºC, and subsequently sown on plates with 0.5x Murashige and Skoog (MS) growth medium (Murashige and Skoog, 1962) (Duchefa Biochemie, Haarlem, The Netherlands) supplemented with 0.1% (w/v) sucrose, 0.05% (w/v) MES (pH 5.8, KOH) and 0.8% (w/v) agar. To grow seedlings devoid of glutathione, seeds were germinated on plates supplemented with 1 mM L-buthionine sulfoximine. For experiments with leaf discs, 5-day-old seedlings were transferred to soil and grown under the same controlled conditions for 4-5 weeks. Leaves were excised using a 7 mm-diameter cork borer. Rosette size was measured on 4-week-old plants using the Leaflab MATLAB script.

Cloning of HyPer7 into a plant expression vector
The HyPer7 sequence including the original nuclear exclusion sequence (NES) was amplified by PCR from pCS2-HyPer7-NES (Pak et al., 2020) using the primers HyPer7_KpnI fw: AGGTACCATGCACCTGG and HyPer7_SalI rv TATGTCGACTTACAGGGTCAGC, which added restriction sites for KpnI and SalI. The fragment was sub-cloned into pJET1.2 (Thermo Fisher Scientific, Waltham, WA), digested with KpnI and SalI (Thermo Fischer Scientific) and purified from an agarose gel with the NucleoSpin ® gel and PCR clean-up kit (Macherey-Nagel, Düren, Germany). For constitutive expression in plants, the fragment was ligated into the vector pBinCM, which is a modified version of the original pBinAR with a UBQ10pro instead of 35Spro (Aller et al., 2013). Plasmid were confirmed by sequencing, transformed into Agrobacterium tumefaciens (AGL1) and used for transformation of Arabidopsis plants by floral dip (Clough and Bent, 1998).

Cloning of HyPer7 into a bacterial expression vector and protein purification
The HyPer7 sequence was amplified by PCR from pCS2-HyPer7-NES (Pak et al., 2020) using the primers HyPer7_gw f: GGGGACAAGTTTGTACAAAAAAGCAGGCTTTCATGCACCTGGCTAATGAGGA and HyPer7_gw_r:GGGGACCACTTTGTACAAGAAAGCTGGGTCCAGGGTCAGCCGCTCCAGGG, which added attB sites for Gateway cloning. The fragment was sub-cloned into pDONR201 and then into the destination vector pETG-10a by recombination using the respective enzymes (Invitrogen, Carlsbad, CA). Escherichia coli (Rosetta strain) cells harboring the pETG-HyPer7-His vector were cultured in liquid LB media supplemented with 100 µg mL -1 ampicillin at 37ºC. Isopropyl-β-D-Thiogalactopyranoside (IPTG) at a final concentration of 1 mM was used for expression induction. Cell lysis was performed as described in (Nietzel et al., 2019) while protein purification was performed as described in (Ugalde et al., 2021).

Confocal laser scanning microscopy and image analysis
Plants and recombinant HyPer7 were imaged in a confocal laser scanning microscope (Zeiss LSM 780, connected to an Axio Observer.Z1; Carl Zeiss Microscopy, Jena, Germany) using a 5x lens (EC Plan-Neofluar 5x/0.16) or a 40x lens (C-Apochromat 40x/1.2 W Korr). Samples were mounted in imaging buffer (10 mM MES, 10 mM MgCl2, 10 mM CaCl2, 5 mM KCl, pH 5.8). All sensors were sequentially excited at 488 nm and 405 nm, and the fluorescence emission was collected at 508-535 nm. The laser power was adjusted according to the respective relative excitation efficiencies on the two excitation peaks of the respective probes. Imaging was performed without averaging with a pixel dwell time of 1.58 µs/pixel. Samples were scanned in areas set to 235.7 µm x 235.7 µm, 124.8 µm x 124.8 µm or 60.7 µm x 60.7 µm. Hydrogen peroxide (H2O2) and dithiothreitol (DTT) perfusion experiments were performed as described in (Ugalde et al., 2020) using a RC-22 perfusion chamber mounted on a P1 platform (Warner Instruments, Hamden, CT). To inhibit photosystem II (PSII), samples were pre-treated for 45 min with 20 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in the dark (Sigma-Aldrich, Steinheim, Germany) dissolved in ethanol. The fluorescence ratio was calculated as 405 nm / 488 nm for roGFP2-Orp1 and 488 nm / 405 nm for HyPer and HyPer7 and normalized to the ratio values at the start of the experiments (t = 0 min). Ratiometric imaging and analysis of single plane images was performed with a custom-written MATLAB script (Fricker, 2016) or FIJI (Schindelin et al., 2012).

Multiwell plate reader measurements
Leaf disks from 4 or 6-week-old plants expressing HyPer7 or 1 µM recombinant HyPer7 were placed in black 96-well plates (Thermo Fischer Scientific). Leaf disks were submerged in 200 µL imaging buffer and the recombinant sensor was diluted in 10 mM Tris-HCl buffer (pH = 7.4). Samples were excited from 362 to 500 nm with a step width of 1 nm and the emission collected at 520±5 nm using a CLARIOstar plate reader (BMG Labtech, Ortenberg, Germany). Complete sensor reduction or oxidation in leaf disks was achieved by supplementing the imaging buffer with 10 mM DTT or 10 mM H2O2, respectively. Non-transformed leaf discs were treated under the same conditions and used for autofluorescence subtraction. For recombinant HyPer7, complete sensor oxidation was reached supplementing the Tris-HCl buffer with 0.1 or 10 mM H2O2. To reduce the sensor, 10 mM DTT or 5 mM Tris(2carboxyethyl)phosphine hydrochloride (TCEP) (Bond-Breaker Solution TM , Thermo Fischer Scientific) were added to the buffer. Time-resolved ratiometric measurements were performed as described in (Ugalde et al., 2021). All sensors were sequentially excited with the filters 400 ± 5 nm and 482 ± 8 nm, and the fluorescence was collected at 520 ± 5 nm. Gains were adjusted to 1900(400 nm) and 1200(482 nm) for roGFP2-Orp1 and 1800(400 nm, 482 nm) for HyPer and HyPer7. The fluorescence ratio was calculated as 400 nm / 482 nm for roGFP2-Orp1 and 482 nm / 400 nm for HyPer and HyPer7. The calculated ratios were normalized to the value at t = 0 h. To induce oxidative stress by endogenous ROS production, either methyl viologen (MV) (Sigma-Aldrich) was added at a final concentration of 50 µM, or the elicitor flg22 (Eurogentec, Seraing, Belgium) was added to a final concentration of 10 µM. After 2 h of incubation in MV, the measurement was paused and samples were exposed to actinic light with a photon flux density of 200 μmol m −2 s −1 for 1 h, after which the fluorescence recordings were resumed. For elicitor-induced ROS production, flg22 was added 20 minutes after start of recording of steady state ratio values.

pH-clamp experiments
To clamp the pH in plants, seedlings were incubated for 1.5 hours in 50 mM NaAc and either 50 mM MES-KOH pH 6.35, 50 mM MOPS-KOH pH 7.35, 50 mM TRICINE-KOH pH 8.35 or 50 mM TRICINE-KOH pH 9.35. Fluorescence measurements were carried on a Leica SP8 inverted laser scanning microscope, with a 40x water objective (HCX Plan Apochromat CS 1.1 NA) and with 2x zoom (273 nm/pixel). For each observation, two images were taken with sequential excitation at 405 nm and 488 nm, corresponding to the two excitation peaks of the fluorescent proteins. Emitted light was collected between 505 and 550 nm.