Diverse subcellular locations of cryptogein-induced reactive oxygen species production in tobacco Bright Yellow-2 cells

Abstract

Reactive oxygen species (ROS) play a crucial role in many cellular responses and signaling pathways, including the oxidative burst defense response to pathogens. We have examined very early events in cryptogein-induced ROS production in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells. Using Amplex Red and Amplex Ultra Red reagents, which report real-time H2O2 accumulation in cell populations, we show that the internal signal for H2O2 develops more rapidly than the external apoplastic signal. Subcellular accumulation of H2O2 was also followed in individual cells using the 2',7'-dichlorofluorescein diacetate fluorescent probe. Major accumulation was detected in endomembrane, cytoplasmic, and nuclear compartments. When cryptogein was added, the signal developed first in the nuclear region and, after a short delay, in the cell periphery. Interestingly, isolated nuclei were capable of producing H2O2 in a calcium-dependent manner, implying that nuclei can serve as a potential active source of ROS production. These results show complex spatial compartmentalization for ROS accumulation and an unexpected temporal sequence of events that occurs after cryptogein application, suggesting novel intricacy in ROS-signaling cascades.

Figure 1.

Generation of H₂O₂ in cryptogein-treated BY-2 cells as reported by AR and AUR. A, AUR assay of cryptogein-treated cells. Left, Representative kinetics graph of H₂O₂ response to cryptogein (100 nm) as measured by fluorescence emission in the absence or presence of DPI (2 μm) and catalase (628 units/mL). Arrow indicates the addition of cryptogein. Left inset, Representative confocal image of cryptogein-treated cells in the presence of AUR reagent. Right, Maximal reaction response rates before and after cryptogein addition (average of five experiments ± sd). B, AR assay of cryptogein-treated cells as described in A. C, Measurement of the kinetics of H₂O₂ accumulation in cryptogein response. Left, Accumulation of H₂O₂ with AR and AUR after the addition (arrow) of cryptogein (n = 5, average ± sd). Right, Calculated time response to achieve maximal linear accumulation after the addition of cryptogein (±sd).

Figure 2.

Subcellular accumulation of H₂O₂ in BY-2 cells after H₂O₂ and cryptogein addition as reported by DCF. A, Left inset, Optical section showing the DCF signal before and 13 min after the addition of 100 nm cryptogein. Right inset, Kinetics graph of response to cryptogein reported by DCF with or without the addition of DPI. B, Subcellular accumulation of H₂O₂. Left insets, DCF signal before and 75 s after external addition of 0.1 mm H₂O₂. Right insets, Image of DCF signal before and 5 min after addition of 100 nm cryptogein. Top insets in B, Division of the cell into periphery (blue line) and nuclear (orange line) regions. Scale bar in bottom right image = 10 μm. C, Kinetics graphs of signal intensity after H₂O₂ (left inset) and cryptogein (right inset) application. DCF signals were recorded from the designated periphery and nuclear subcellular compartments as shown in the top insets of B. D, Maximal rate of signal acquisition as measured in the periphery or nucleus expressed as the time gap. The time to achieve maximal rate of signal acquisition was measured in each compartment. The difference in start times was calculated as described in “Materials and Methods.” Negative values were defined when the nuclear compartment reacted before the periphery compartment. Averages of five experiments ± se are shown.

Figure 3.

Imaging of H₂O₂ and subcellular-specific stains. A, Optical section of cells double stained with DCF and the membrane-specific marker FM 4-64. B, Optical section of cells double stained with DCF and mitochondrial-specific marker MitoTracker. C, Optical section of cells double stained with DCF and the ER-specific marker DPX. Right insets in each image are enlarged sections of the cell periphery (top inset) and the nuclear regions (bottom inset). Scale bars in bottom image = 10 μm.

Figure 4.

Imaging of H₂O₂ and subcellular-specific stains in BFA-treated BY-2 cells. A, Cells were stained separately with the ER marker DiOC₅(3) or DCF. Left insets, 3D reconstructions of cells stained with DiOC₅(3). Right insets, 3D reconstructions of cells stained with DCF. Images of nontreated cells (control; top insets) and between 30 and 90 min of BFA treatment (bottom insets) were collected. B, Cells were double stained with DCF and the ER-specific marker DPX. Top inset, Nontreated cells showing DCF, DPX, and merge images; bottom insets, DCF, DPX, and merge images were collected at 30 min (middle inset) and 60 min (bottom inset) after BFA addition. Scale bar = 10 μm.

Figure 5.

Imaging of H₂O₂ signal in BY-2 cells and isolated nuclei in response to calcium application, as reported by DCF and AUR. A, Merged optical section of isolated BY-2 nucleus triple stained with FM 4-64, DAPI, and DCF. B, Optical section of cells stained with DCF before and after the addition of 1 mm calcium. C, Optical section of isolated nuclei stained with DCF (top inset) and AUR (bottom inset) before (control) and after the addition of 1 mm calcium (+Ca²⁺), and transmission images of the isolated nuclei. Scale bar = 10 μm. D, Representative graph of calcium-dependent DCF response in isolated nuclei. E, Reaction rates after 1 mm calcium addition (average of at least five experiments ± sd).