Production of reactive oxygen species and wound-induced resistance in Arabidopsis thaliana against Botrytis cinerea are preceded and depend on a burst of calcium

Abstract

Background: Wounded leaves of Arabidopsis thaliana produce reactive oxygen species (ROS) within minutes after wounding and become resistant to the pathogenic fungus Botrytis cinerea at a local level. This fast response of the plants to the wound is called wound-induced resistance (WIR). However the molecular mechanisms of this response and the signal cascade between the wound and ROS production are still largely unknown. Calcium is a conserved signal and it is involved in many abiotic stress responses in plants, furthermore, calcium pathways act very fast.

Results: The results of this study show that leaves treated with calcium channels inhibitors (verapamil) or calcium chelators (oxalate and EGTA) are impaired in ROS production. Moreover, leaves treated with verapamil, EGTA or oxalate were more susceptible to B. cinerea after wounding. The intracellular measurements of calcium changes indicated quick but transient calcium dynamics taking place few seconds after wounding in cells neighbouring the wound site. This change in the cytosolic calcium was followed in the same region by a more stable ROS burst.

Conclusions: These data further extend our knowledge on the connection between wounding, calcium influx and ROS production. Moreover they provide for the first time the evidence that, following wounding, calcium changes precede a burst in ROS in the same location.

Figure 1

Treatment of A. thaliana leaves with calcium channel blocker and calcium chelators abolishes ROS production after leaves wounding. Droplets containing different concentrations of verapamil (10 μM, 100 μM, 1 mM, 10 mM), EGTA (1 mM, 10 mM, 50 mM, 100 mM) and oxalate (100 μM, 1 mM, 10 mM, 50 mM) were applied on leaves for 3 hours. Leaves were then either wounded and stained with DCF-DA and visualized at a fluorescence microscope with two sets of filters (ROS in green and chlorophyll autofluorescence in red, upper panels), or were tested for cell viability using trypan blue (lower panels). Densitometric analysis of the ROS signal is displayed on the right of each image series. Asterisks represent significant differences using Student's t test relative to water-treated control; *P < 0.05, **P < 0.01.

Figure 2

Calcium channel blocker and calcium chelators abolish WIR. Droplets containing different concentrations of verapamil (10 μM, 100 μM, 1 mM, 10 mM), EGTA (1 mM, 10 mM, 50 mM, 100 mM) and oxalate (100 μM, 1 mM, 10 mM, 50 mM) were applied on leaves for 3 hours. After droplet removal, leaves were wounded to induce WIR and inoculated with B. cinerea spores on the same site where inhibitors were applied. After three days of incubation in covered trays, lesion diameters were measured. Black: non-wounded leaves. Grey: leaves wounded right after inhibitors removal to induce WIR. Asterisks represent significant differences using Student's t test relative to water-treated control; *P < 0.05, **P < 0.01.

Figure 3

Calcium is accumulated in the cytosol after wounding. Leaves that constitutively express cytosolic aequorin are incubated over-night in 10 μM CTZ, placed into a luminometer and then wounded. Luminescence was measured and depended on the amount of calcium that binds to aequorin and thus reflects cytosolic calcium influx. In the control, leaves were infiltrated with water, otherwise with the mentioned concentrations of calcium inhibitors.

Figure 4

Kinetics of cytosolic calcium influx after wounding. Calcium appearance in cytosol of A. thaliana leaves after wounding was measured with plants expressing Cameleon YC3.6 infiltrated with water or with 100 mM EGTA. Upper panels: microscope images display key time points on the time course. Lower panels: kinetics resulting from the quantification of the fluorescent signals (black: interveinal tissue, red: vein). The experiments were repeated five times giving comparable kinetics, one representative time-course is presented.

Figure 5

ROS and calcium accumulate in the same cells with different kinetics. (A) Redox potential after wounding of A. thaliana leaves is measured with roGFP2 expressing plants. Upper panels: microscope images display key time points on the time course. Lower panels: kinetics resulting from the quantification of the fluorescent signals. (B) Leaf discs obtained from plants constitutively expressing YC3.6 protein were infiltrated with DCF-DA and gently wounded with a yellow tip. FRET efficiency (upper panel) and ROS formation (lower panel) were scored in a time course with a confocal microscope taking a set of pictures every minute. The experiments were repeated 10 times giving comparable kinetics, one representative time-course is presented.