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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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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.
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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.

Mentions: Changes in intracellular calcium were further examined using an A. thaliana line expressing the Yellow Cameleon 3.6 fluorescence resonance energy transfer-based Ca2+ sensor in the cytosol [9,10]. Wounding resulted in a first transient and steep increase in the YFP/CFP ratio (from 0 to 2s after wounding), followed by a second, smaller but more sustained one (ca 30s after wounding) (Figure 4). The increase in calcium was restricted to cells proximal to the wound site and was observed in cells composing both epidermis and vascular tissue. Both calcium peaks were abolished in the interveinal tissue of the epidermis when leaves were treated with 100 mM EGTA (black lines and arrows). Surprisingly, only the second calcium peak was abolished in the veins of EGTA-treated leaves (Figure 4). The use of plants expressing the redox sensitive biosensor roGFP2 [11] allowed also to monitor the effect of wounding on the cytosolic redox potential. Wounding resulted in an immediate increase of probe oxidation in cell neighbouring damaged tissue (Figure 5A), reflecting an effect clearly related to the observed production of ROS (Figure 1). The oxidation of roGFP2 reached a maximum after 10–12 minutes after wounding (Figure 5A). After this time it was possible to observe a slower decrease of probe oxidation, probably due to the fact that the damaged leaf is not kept in humid conditions due to equipment limitations. An additional effort was made to co-localize the calcium signal with the generation of ROS after wounding. Leaf discs of YC3.6-expressing leaves were infiltrated with the ROS-sensitive probe DCF-DA prior to observation with confocal laser scanning microscope. The time course of changes in calcium and ROS after wounding is presented in Figure 5B. Wounding lead to a rapid emission of a calcium signal at the wound site (in yellow-red, upper part Figure 5B) that decreased rapidly with time. The appearance of fluorescence (in green, lower part Figure 5B) caused by the sensing of ROS by the DCF-DA probe was also localized at the wound site but took place after the calcium signal. These results indicate that changes in calcium appear earlier than ROS and both occur at the wounding site.


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)

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

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

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.
Mentions: Changes in intracellular calcium were further examined using an A. thaliana line expressing the Yellow Cameleon 3.6 fluorescence resonance energy transfer-based Ca2+ sensor in the cytosol [9,10]. Wounding resulted in a first transient and steep increase in the YFP/CFP ratio (from 0 to 2s after wounding), followed by a second, smaller but more sustained one (ca 30s after wounding) (Figure 4). The increase in calcium was restricted to cells proximal to the wound site and was observed in cells composing both epidermis and vascular tissue. Both calcium peaks were abolished in the interveinal tissue of the epidermis when leaves were treated with 100 mM EGTA (black lines and arrows). Surprisingly, only the second calcium peak was abolished in the veins of EGTA-treated leaves (Figure 4). The use of plants expressing the redox sensitive biosensor roGFP2 [11] allowed also to monitor the effect of wounding on the cytosolic redox potential. Wounding resulted in an immediate increase of probe oxidation in cell neighbouring damaged tissue (Figure 5A), reflecting an effect clearly related to the observed production of ROS (Figure 1). The oxidation of roGFP2 reached a maximum after 10–12 minutes after wounding (Figure 5A). After this time it was possible to observe a slower decrease of probe oxidation, probably due to the fact that the damaged leaf is not kept in humid conditions due to equipment limitations. An additional effort was made to co-localize the calcium signal with the generation of ROS after wounding. Leaf discs of YC3.6-expressing leaves were infiltrated with the ROS-sensitive probe DCF-DA prior to observation with confocal laser scanning microscope. The time course of changes in calcium and ROS after wounding is presented in Figure 5B. Wounding lead to a rapid emission of a calcium signal at the wound site (in yellow-red, upper part Figure 5B) that decreased rapidly with time. The appearance of fluorescence (in green, lower part Figure 5B) caused by the sensing of ROS by the DCF-DA probe was also localized at the wound site but took place after the calcium signal. These results indicate that changes in calcium appear earlier than ROS and both occur at the wounding site.

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