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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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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.
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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.

Mentions: A distinctive feature of WIR is that wounded A. thaliana leaves become resistant to the necrotrophic fungus B. cinerea[5]. Thus, we tested if verapamil and chelating agents affected WIR to B. cinerea. Droplets of tested molecules were applied on leaves and after 3 hours, droplets were removed, leaves were wounded and suspensions of B. cinerea spores were placed on the wound sites. After 3 days of incubation, the wounded control was fully protected while unwounded water-treated controls displayed the typical symptoms of B. cinerea infection (necrosis at the infection site) (Figure 2). Similar to their effect on wound-induced ROS formation, verapamil (starting at 1 mM), EGTA (starting at 10 mM) and oxalate (starting at 10 mM) all affected WIR (Figure 2). Plants treated with oxalate and EGTA without wounding showed an enhanced lesion size, indicating a possible involvement of calcium in the basal resistance (Figure 2). Taken together, these results indicate that calcium might be involved in the pathway that couples perception of wounding with the induction of ROS and WIR.


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)

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

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

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.
Mentions: A distinctive feature of WIR is that wounded A. thaliana leaves become resistant to the necrotrophic fungus B. cinerea[5]. Thus, we tested if verapamil and chelating agents affected WIR to B. cinerea. Droplets of tested molecules were applied on leaves and after 3 hours, droplets were removed, leaves were wounded and suspensions of B. cinerea spores were placed on the wound sites. After 3 days of incubation, the wounded control was fully protected while unwounded water-treated controls displayed the typical symptoms of B. cinerea infection (necrosis at the infection site) (Figure 2). Similar to their effect on wound-induced ROS formation, verapamil (starting at 1 mM), EGTA (starting at 10 mM) and oxalate (starting at 10 mM) all affected WIR (Figure 2). Plants treated with oxalate and EGTA without wounding showed an enhanced lesion size, indicating a possible involvement of calcium in the basal resistance (Figure 2). Taken together, these results indicate that calcium might be involved in the pathway that couples perception of wounding with the induction of ROS and WIR.

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