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

Beneloujaephajri E, Costa A, L'Haridon F, Métraux JP, Binda M - BMC Plant Biol. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of biology, University of Fribourg, Ch, du Musée 10, 1700 Fribourg, Switzerland. matteo.binda@unifr.ch.

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.

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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.
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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.

Mentions: Wounded A. thaliana leaves generate ROS within minutes as revealed after infiltration with the 5-(and-6)-carboxy-2,7-dichlorodihydrofluoresceindiacetate (DCF-DA) probe [5] (Figure 1). We have used a pharmacological approach to examine the importance of calcium in wound-induced ROS formation. A calcium channel blocker (verapamil) or calcium chelators (EGTA or oxalate) were applied as small droplets on the upper side of Arabidopsis leaves. After 3 hours the droplets were removed and leaves were wounded at those sites. Verapamil (starting at 10 μM), EGTA (starting at 50 mM) and oxalate (starting at 50 mM) all affected wound induced ROS formation (Figure 1). Using this way of application, we cannot know how much of these compounds penetrated in the extracellular space. The effects of the chelators were visible at concentrations in the same range of the estimate extracellular calcium concentration (1–10 mM) [7]. A dose–response effect of the compounds tested could be detected starting at lower concentrations only when applied by vacuum-infiltration of detached leaves prior to wounding (Additional file 1). None of these compounds affected cell vitality at the concentrations used as determined using trypan blue tests (Figure 1).


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

Beneloujaephajri E, Costa A, L'Haridon F, Métraux JP, Binda M - BMC Plant Biol. (2013)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016300&req=5

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.
Mentions: Wounded A. thaliana leaves generate ROS within minutes as revealed after infiltration with the 5-(and-6)-carboxy-2,7-dichlorodihydrofluoresceindiacetate (DCF-DA) probe [5] (Figure 1). We have used a pharmacological approach to examine the importance of calcium in wound-induced ROS formation. A calcium channel blocker (verapamil) or calcium chelators (EGTA or oxalate) were applied as small droplets on the upper side of Arabidopsis leaves. After 3 hours the droplets were removed and leaves were wounded at those sites. Verapamil (starting at 10 μM), EGTA (starting at 50 mM) and oxalate (starting at 50 mM) all affected wound induced ROS formation (Figure 1). Using this way of application, we cannot know how much of these compounds penetrated in the extracellular space. The effects of the chelators were visible at concentrations in the same range of the estimate extracellular calcium concentration (1–10 mM) [7]. A dose–response effect of the compounds tested could be detected starting at lower concentrations only when applied by vacuum-infiltration of detached leaves prior to wounding (Additional file 1). None of these compounds affected cell vitality at the concentrations used as determined using trypan blue tests (Figure 1).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of biology, University of Fribourg, Ch, du Musée 10, 1700 Fribourg, Switzerland. matteo.binda@unifr.ch.

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.

Show MeSH
Related in: MedlinePlus