Live Cell Imaging with R-GECO1 Sheds Light on flg22- and Chitin-Induced Transient [Ca(2+)]cyt Patterns in Arabidopsis

Abstract

Intracellular Ca(2+) transients are an integral part of the signaling cascade during pathogen-associated molecular pattern (PAMP)-triggered immunity in plants. Yet, our knowledge about the spatial distribution of PAMP-induced Ca(2+) signals is limited. Investigation of cell- and tissue-specific properties of Ca(2+)-dependent signaling processes requires versatile Ca(2+) reporters that are able to extract spatial information from cellular and subcellular structures, as well as from whole tissues over time periods from seconds to hours. Fluorescence-based reporters cover both a broad spatial and temporal range, which makes them ideally suited to study Ca(2+) signaling in living cells. In this study, we compared two fluorescence-based Ca(2+) sensors: the Förster resonance energy transfer (FRET)-based reporter yellow cameleon NES-YC3.6 and the intensity-based sensor R-GECO1. We demonstrate that R-GECO1 exhibits a significantly increased signal change compared with ratiometric NES-YC3.6 in response to several stimuli. Due to its superior sensitivity, R-GECO1 is able to report flg22- and chitin-induced Ca(2+) signals on a cellular scale, which allowed identification of defined [Ca(2+)]cyt oscillations in epidermal and guard cells in response to the fungal elicitor chitin. Moreover, we discovered that flg22- and chitin-induced Ca(2+) signals in the root initiate from the elongation zone.

Keywords: Arabidopsis; R-GECO1; calcium imaging; chitin; flg22; sensor.

Figure 1. R-GECO1 Exhibits Enhanced Ca²⁺-Dependent Signal Change Compared with NES-YC3.6

Ca²⁺-dependent signal changes in response to 1 mM ATP in roots of 6- to 8-day-old seedlings expressing NES-YC3.6 and R-GECO1. (A) Fluorescence images of ECFP, cpVenus, R-GECO1, and corresponding bright field image. Scale bar represents 50 μm. (B) Time-dependent fluorescence intensities. (C) Time-dependent normalized NES-YC3.6 emission ratios (ΔR/R) and normalized R-GECO1 fluorescence intensities (ΔF/F). (D–F) Maximum signal change (D), signal-to-noise ratios (E), and SD of the baseline (F). Error bars represent SD of three independent experiments.

Figure 2. Visualization of Tip-Localized [Ca²⁺]_cyt Gradients

(A and B) Tip-localized [Ca²⁺]_cyt gradients were visualized in germinating pollen tubes (A) and growing root hair cells (B) expressing NES-YC3.6 and R-GECO1. Shown are ratiometric images for NES-YC3.6, fluorescence images for R-GECO1, and the corresponding bright field images. (C) Time-dependent [Ca²⁺]_cyt dynamics in a growing root hair. Fluorescence images of R-GECO1 at different time points. Time format, sss. (D) Time-dependent normalized R-GECO1 fluorescence intensities (ΔF/F) were extracted from the apex of a growing root hair indicated by the circled area in (C). Arrowheads in (D) correspond to the images shown in (C). Scale bars in (A–C) represent 20 μm.

Figure 3. R-GECO1 Is More Sensitive toward Changes of pH than NES-YC3.6

pH-dependent signal changes in response to pH equilibration buffers in roots of 6- to 8-day-old seedlings expressing pHGFP or NES-YC3.6 and R-GECO1. For cytosolic pH adjustment, pH equilibration buffers ranging from pH 6.8–8.0 were applied sequentially to the seedlings. To suppress Ca²⁺ ion fluxes during the pH treatments, indicated concentrations of LaCl₃, EGTA, and BAPTA-AM were applied simultaneously. (A) Fluorescence image of pHGFP and the corresponding bright field image. (B) Time-dependent normalized pHGFP ratio changes (ΔR/R). (C) Fluorescence images of ECFP, cpVenus, R-GECO1, and corresponding bright field image. (D) Time-dependent fluorescence intensities of ECFP, cpVenus, and R-GECO1. (E) Time-dependent normalized NES-YC3.6 emission ratios (ΔR/R) and normalized R-GECO1 fluorescence intensities (ΔF/F). Data are representative of n = 5 measurements. Scale bars in (A) and (C) represent 50 μm.

Figure 4. R-GECO1 Detects PAMP-Triggered [Ca²⁺]_cyt Oscillations in Intact Leaves

Ca²⁺-dependent signal changes in response to flg22 (A, B) and chitin (C, D) in true leaves of 14- to 16-day-old seedlings expressing NES-YC3.6 and R-GECO1. Fluorescence images of R-GECO1 5:00 min after flg22 (A) and 11:17 min after chitin application (C). (B, D) Time-dependent normalized NES-YC3.6 emission ratios (ΔR/R) and normalized R-GECO1 fluorescence intensities (ΔF/F) calculated from ROIs 1–4 indicated in (A) and (C). Shown are representative experiments with n ≥ 6. Scale bars in (A) and (C) represent 50 μm and scale bars in (B) and (D) indicate ΔF/F (R-GECO1) and DR/R (NES-YC3.6) with y = 1.

Figure 5. Ca²⁺ Signaling in Response to flg22 and Chitin in Guard Cells and Epidermal Cells

Ca²⁺-dependent signal changes in response to flg22 and chitin in true leaves of 14- to 16-day-old seedlings were measured with different experimental setups. (A–D) Fluorescence images of R-GECO1. (A) Top imaging 3:10 min after flg22 application. (B) Top imaging 4:15 min after chitin application. (C) Bottom imaging 12:25 min after flg22 application. (D) Bottom imaging 9:52 min after chitin application. (E–H) Graphs show time-dependent normalized R-GECO1 fluorescence intensities (ΔF/F) calculated from ROIs 1–8 outlined in (A–D). Different treatments are indicated by gray boxed areas. (E) Top imaging 100 nM flg22. (F) Bottom imaging 100 nM flg22. (G) Top imaging 100 μg/ml chitin. (H) Bottom imaging 100 μg/ml chitin. (I) Percentage of guard cells that exhibited significant [Ca²⁺]_cyt elevations in response to 100 nM flg22 (n = 66) or 100 μg/ml chitin (n = 74) as revealed in the top imaging setup. (J) Percentage of guard cells that exhibited significant [Ca²⁺]_cyt elevations in response to 100 nM flg22 (n = 37) or 100 μg/ml chitin (n = 34) as revealed in the bottom imaging setup. (A–H) Shown are representative experiments with n ≥ 6. Scale bars in (A–D) represent 15 μm and scale bars in (E–H) indicate ΔF/F with y = 1.

Figure 6. Flg22- and Chitin-Induced [Ca²⁺]_cyt Transients in the Root Originate from the Elongation Zone

Ca²⁺-dependent signal changes in response to 1 μM flg22 (A, C, E) and 100 μg/ml chitin (B, D, F) in roots of 6- to 7-day-old seedlings. Ca²⁺ imaging was performed in the RootChip16. (A and B) Time series of normalized R-GECO1 fluorescence images (ΔF/F). (C and D) Kymographs were extracted along three pixel-wide dashed lines indicated in (A) and (B). (E and F) Normalized R-GECO1 fluorescence intensities (ΔF/F) were measured from ROIs in the elongation and the root hair zone indicated by the boxed areas in (A) and (B). Shown are [Ca²⁺]_cyt dynamics in response to flg22 and chitin of three independent roots. Arrowheads correspond to the images shown in (A) and (B). Gray boxes in (C–F) indicate a 5-min square pulse of flg22 or chitin, respectively. Scale bars in (A) and (B) indicate 200 μm and scale bars in (E) and (F) indicate ΔF/F with y = 0.1. Time format, m:ss. Note that the apparent increase in signal at the root cap in (A) and (B) does not indicate high levels of [Ca²⁺]_cyt. It is a result of image calculation due to the growth of the root. This area was not included in the analysis.