Hydrogen peroxide sensor HyPer7 illuminates tissue-specific plastid redox dynamics

Abstract

The visualization of photosynthesis-derived reactive oxygen species has been experimentally limited to pH-sensitive probes, unspecific redox dyes, and whole-plant phenotyping. Recent emergence of probes that circumvent these limitations permits advanced experimental approaches to investigate in situ plastid redox properties. Despite growing evidence of heterogeneity in photosynthetic plastids, investigations have not addressed the potential for spatial variation in redox and/or reactive oxygen dynamics. To study the dynamics of H2O2 in distinct plastid types, we targeted the pH-insensitive, highly specific probe HyPer7 to the plastid stroma in Arabidopsis (Arabidopsis thaliana). Using HyPer7 and glutathione redox potential (EGSH) probe for redox-active green fluorescent protein 2 genetically fused to the redox enzyme human glutaredoxin-1 with live cell imaging and optical dissection of cell types, we report heterogeneities in H2O2 accumulation and redox buffering within distinct epidermal plastids in response to excess light and hormone application. Our observations suggest that plastid types can be differentiated by their physiological redox features. These data underscore the variation in photosynthetic plastid redox dynamics and demonstrate the need for cell-type-specific observations in future plastid phenotyping.

Figure 1.

Expression and targeting of SSU:HyPer7. A–C) Stable expression of UBQ10:SSU:HyPer7 in an A. thaliana (Col-0) true leaf excited by its 2 relative maxima, 488 and 405 nm and collected between 505 and 550 nm. Whole-leaf scanning was performed on a Zeiss LSM-510 Meta fitted with a 10× Epiplan-Neoflaur objective using the tile scan feature in Zen software. D to G) HyPer7 (collected between 505 and 550 nm), excited with a 488 nm Argon Laser and imaged with a 40× c-Plan-Apochromat objective colocalizes with chlorophyll autofluorescence (650 nm LP) in abaxial pavement cells of 8-d-old cotyledons. H to K) Max intensity projection Z-stack shows HyPer7 (Excitation, 488 nm; emission, 505 to 550 nm) localizing within stromules (white arrow). White box in H) denotes the magnified J and K). Z-stack was filtered with a 1-pixel x, y, z radius median filter prior to max intensity projection.

Figure 2.

HyPer7 reports light-dependent stromal H₂O₂ at large dynamic range. Ratiometric HyPer7 micrographs cropped using segmented plastid masks treated with either 10 mM H₂O₂ A), Untreated B), or 10 mM DTT C) for 30 min prior to imaging. D) Estimation of HyPer7 dynamic range following 30-min incubation with either 10 mM H₂O₂ or 10 mM DTT. White triangle denotes the per-plastid mean from multiple individual plants (N > 120 plastids per treatment). Individual points reflect individual segmented plastids, box indicates interquartile range with center line indicating treatment median. Lines extending beyond box indicate 1.5× interquartile range. E) Mean HyPer7 ratio, per plastid, ±1 Se from abaxial pavement cell sensory plastids imaged within 2 h of subjective dawn at indicated age and tissue. Line indicates generalized additive model ±Se fitted to each developmental stage. N > 250 plastids at each time point derived from 6 to 8 different leaves or cotyledons. Data for 2-wk-old true leaf oxidation curves were taken from the same experiment as presented in G). F) HyPer7 oxidation status relative to the first frame average (R-R′) at end of imaging time course. Letters indicate significantly different groups (Tukey-HSD test, P < 0.01). Sample means are indicated with white triangles. Data were taken from the same experiment as presented in E). Individual points reflect individual segmented plastids, box indicates interquartile range with center line indicating treatment median. Lines extending beyond box indicate 1.5× interquartile range. G) Per plastid, mean 405 or 488 channel intensities ±1 Se over the imaging time course from first and second true leaves of 2-wk-old seedlings presented in E). HyPer7 oxidation in guard cell plastids before H) and after varied 488 nm laser output using ROI + bleaching feature on LSM510. Representative micrographs of HyPer7 488/405 ratios prior to I) and post J) excitation of guard cell chloroplasts with varying laser intensities. Asterisks overlaid on micrographs in J) indicate percent laser output used for 50 iterations of excitation with 488 nm laser at each ROI; ****10%, ***5%, **0.5%, and *0%. Imaging in H), I), and J) was performed with settings used to minimize laser output and exposure time by opening the confocal pinhole to 2.2 AU on an LSM510-Meta and collecting fluorescence at 505 to 550 nm using a 20×/0.8NA Plan-Apochromat objective at 3× digital zoom, 488 nm at 0.2% output, and 405 at 2.5% output with a single average (exposure time 1.9 s). H) Quantification of HyPer7 488/405 ratio relative to the pretreatment average. ***P < 0.001, ****P <<< 0.001, Wilcoxon ranked sums test, n > 12 plastid foci derived from 4 guard cell pairs on fully expanded true leaves collected at bolting. Data are relative (R-R′) to the average preincubation ratio. White triangle denotes sample mean. Individual points reflect individual segmented plastids, box indicates interquartile range with center line indicating treatment median. Lines extending beyond box indicate 1.5× interquartile range. K) HyPer7 488/405 ratio in abaxial sensory plastids from 8-d-old seedling cotyledons relative (R-R′) to pretreatment sample average before or after 10-min incubation with brightfield halogen lamp at varying output. **P < 0.01 Wilcoxon ranked sums test, n > 100 plastids derived from 7 independent leaves. White triangle indicates sample mean. Individual points reflect individual segmented plastids, box indicates interquartile range with center line indicating treatment median. Lines extending beyond box indicate 1.5× interquartile range. Abbreviations: ROI, region of interest; AU, arbitrary units; DTT, dithiothreitol.

Figure 3.

HyPer7 remains specific to H₂O₂, in vivo. A and B) Representative ratiometric images at the outset and end of imaging time course for seedlings pretreated with either 1 mM l-ascorbate A) or 1 mM NaCl B). Scale bar indicated in A) applies to all images as they were acquired with identical settings. C) Quantification of stromal HyPer7 oxidation in seedlings pretreated with either 1 mM AsA or 1 mM NaCl (osmotic control) prior to imaging stress ****P <<< 0.0001 (Wilcoxon ranked sum test). White triangle indicates per-plastid mean. White triangle indicates sample mean. Individual points reflect individual segmented plastids, box indicates interquartile range with center line indicating treatment median. Lines extending beyond box indicate 1.5× interquartile range. D) Mean HyPer7 ratio, per plastid, ±1 Se relative to the first frame from abaxial pavement cell sensory plastids over an imaging time course. Seedlings were vacuum infiltrated with a syringe and incubated with either 1 mM AsA or 1 mM NaCl (at pH = 5.7) as osmotic control for 30 min prior to imaging. N > 97 plastids coming from multiple independent plants imaged within 2 h of subjective dawn. Data are from the same experiment presented in C). E) Quantification of stromal HyPer7 oxidation in seedlings pretreated with either 20 µM DCMU or mock (DMSO) prior to imaging stress (N > 200 plastids, **P < 0.01, Wilcoxon ranked sum test). White triangle indicates sample mean. Individual points reflect individual segmented plastids, box indicates interquartile range with center line indicating treatment median. Lines extending beyond box indicate 1.5× interquartile range. F) Mean HyPer7 ratio, per plastid, ±1 Se relative to the first frame from abaxial pavement cell sensory plastids over an imaging time course. Seedlings were pretreated with either 20 µM DCMU or mock (DMSO) and imaged within 2 h of subjective dawn. Data are from the same experiment presented in E). Abbreviations: AsA, L-ascorbate; AU, arbitrary units; DCMU, N-(3,4-dichlorophenyl)-N-dimethylurea.

Figure 4.

HyPer7 reports cell-type heterogeneity in imaging-dependent H₂O₂ production. A) Ratiometric images of stromal HyPer7 oxidation over imaging time course. Images were obtained by dividing, pixel-by-pixel a 1-pixel-radius median-filtered 488/405 channels. Plastids were cropped using a binary mask obtained by thresholding the 488 channel. B) Stromal HyPer7 oxidation prior to excess-light stress compared across cell types of 8-d-old seedlings grown on 1/2 MS media at subjective dawn. Significantly different groups were determined using a Tukey-HSD test with P-value cutoff of 0.01 (N > 55, 500, 100, and 87 for guard cell, pavement cell, petiole epidermis, and root plastids, respectively). White triangle indicates sample mean. Individual points reflect individual segmented plastids, box indicates interquartile range with center line indicating treatment median. Lines extending beyond box indicate 1.5× interquartile range. C) Stromal HyPer7 oxidation prior to excess-light stress compared across epidermal cell types of overnight-equilibrated leaf discs from Col-0 collected at bolting. Significance denoted by letters, Tukey-HSD test with P-value cutoff of 0.01 (N > 160 and 255 for guard cell plastids and pavement cell plastids, respectively). White triangle indicates sample mean. Individual points reflect individual segmented plastids, box indicates interquartile range with center line indicating treatment median. Lines extending beyond box indicate 1.5× interquartile range. D and E) Relative stromal HyPer7 oxidation over the imaging time course separated by cell type in D) 8-d-old seedlings or E) equilibrated leaf discs. Data are from the same experiments presented in B and C). Line indicates generalized additive model ±Se fitted to each cell type. F) Stromal oxidation of GRX1-roGFP2 in 8-d-old seedlings separated by cell type. Significantly different groups were determined using a Tukey-HSD test with P-value cutoff of 0.001 (N = 73, 192, and 81 for guard cell, pavement cell, and petiole epidermis, respectively). White triangle indicates sample mean. Individual points reflect individual segmented plastids, box indicates interquartile range with center line indicating treatment median. Lines extending beyond box indicate 1.5× interquartile range. G) Representative ratiometric images of stromal GRX1-roGFP2 oxidation in 8-d-old seedlings at subjective dawn. Images were obtained by dividing, pixel-by-pixel, 405/488 channels filtered with a 1-pixel radius Gaussian filter. Plastids were cropped using a binary mask obtained by thresholding a median-filtered 488 channel. Abbreviations: AU, arbitrary units.

Figure 5.

Stromal H₂O₂ accumulates independent of photosystems in response to ABA. A and B) Initial stromal oxidation of 8-d-old cotyledon abaxial pavement cell A) or guard cell B) plastids in response to 15 min dark pretreatment with 20 µM ABA shortly after subjective dawn. White triangle indicates mean (55 < N < 380 plastids from multiple independent plants, ****P << 0.001 and **P < 0.01, Wilcoxon ranked sums test). Individual points reflect individual segmented plastids, box indicates interquartile range with center line indicating treatment median. Lines extending beyond box indicate 1.5× interquartile range. C) Representative micrographs of guard cell plastids with pseudo-colored stromal HyPer7 488/405 ratio. 488- and 405-nm channels were filtered with 1-pixel radius Gaussian filter prior to ratio calculation. Relative (R-R′) oxidation of pavement cell D) or guard cell E) stromal HyPer7 ±Se over excess imaging light treatment following dark pretreatment with 20 µM ABA or solvent control. Data are from same experiments presented in A and B). Trend line indicates generalized additive model ±Se. F) Stromal HyPer7 oxidation status of 8-d-old cotyledon guard cells following 1 h dark pretreatment with 20 µM DCMU or DSMO and subsequent 20 µM ABA spike-in for 15 min prior to imaging. Letters indicate significantly different groups determined using a Tukey-HSD test with P-value cutoff of 0.01, n > 50 plastids from at least 5 independent plants. White triangle indicates sample mean. Individual points reflect individual segmented plastids, box indicates interquartile range with center line indicating treatment median. Lines extending beyond box indicate 1.5× interquartile range. G) Relative (R-R′) stromal HyPer7 488/405 ratio ±Se over imaging time course in 8-d-old cotyledons guard cells in the presence of DCMU and/or ABA. Trend lines indicate generalized additive model ±Se. Data are from the same experiments presented in F). H) Representative pseudo-colored micrographs of HyPer7 488/405 ratio in guard cell plastids. 488- and 405-nm channels were filtered with 1-pixel radius Gaussian filter prior to ratio calculation. I) Comparison of HyPer7 stromal oxidation in guard cells pretreated with DCMU and spiked-in with 20 µM ABA or solvent control in 2-wk-old first and second true leaves. Triangles indicate mean relative (R-R^mock) HyPer7 oxidation (N > 160 plastids from multiple independent plants, ****P << 0.001, Wilcoxon ranked sums test). Individual points reflect individual segmented plastids, box indicates interquartile range with center line indicating treatment median. Lines extending beyond box indicate 1.5× interquartile range. Abbreviations: ABA, abscisic acid; AU, arbitrary units; DCMU, N-(3,4-dichlorophenyl)-N-dimethylurea.