Effect of Calcium and Calcium-Counteracting Drugs ...

Plant CellPhysioI. 38(6): 751-753 (1997) JSPP © 1997

Short Communication

Effect of Calcium and Calcium-Counteracting Drugs on the Response of Bidens pilosa L. to Wounding Alain Vian 1 - 2 - 56 , Chantal Henry-Vian 1 2 - 5 , Rodolphe Schantz3, Marie-Luce Schantz3, Eric Davies 4 , Gerard Ledoigt 2 and Marie-Odile Desbiez' 1

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Physiologie Integree de I'Arbre Fruitier, Unite associee INRA/Universite Blaise Pascal, Campus Universitaire des Cezeaux, 63177 Aubiere, France Organisation et Variabilite du Genome Vegetal, Unite associee INRA/Universite Blaise Pascal, Campus Universitaire des Cezeaux, 63177 Aubiere, France Institut de Biologie Moleculaire des Plantes, CNRS, 12 rue du General Zimmer, 67084 Strasbourg Cedex, France North Carolina State University, Department of Botany, BOX 7612, Raleigh NC 27695, U.S.A.

We used calcium counteracting drugs known to reduce the amplitude of wound-induced electric wave of depolarization and we showed that in these conditions, accumulation of the calmodulin mRNA (recently found to be correlated to membrane potential) is strongly reduced. These results bring additional evidence linking membrane potential and calmodulin mRNA accumulation. Key words: Bidens pilosa — Calcium — Calmodulin — Signal transmission — Wounding.

Plants can perceive local injurious stimuli, such as wounding, naming or crushing, that are transduced into an informative signal propagated throughout the whole plant, thereby evoking a systemic response. For example, wounding, application of jasmonic acid and electrical stimulation of solanaceous plants evoke a systemic accumulation of the proteinase inhibitor pin-2 transcripts (Pena-Cortes et al. 1995). The exact nature of the informative signal is still in debate, while both chemical (abscisic acid, systemin, oligosaccharides) and physical agents (hydraulic signal, membrane potential) may be part of it (Davies 1993, Stankovic and Davies 1996). In the composite Bidens pilosa, an injurious stimulus (wound, heat) given to the cotyledons results in rapid metabolic changes in the hypocotyl (Desbiez et al. 1983, HenryVian et al. 1995a, b). The metabolic changes observed in the intact, distant hypocotyl imply the transmission of a traumatic signal. These stimuli elicite an electrical wave of depolarization named Slow Wave (SW) or Variation Potential (VP), which has been extensively studied (Julien et al. 1991), and proposed to be part of the signalling mechanism Abbreviations: CaM, Calmodulin; EGTA, ethyleneglycolbis-(/S-aminoethylether)yV,yV,A^7V-tetraacetic acid. 3 Present address: North Carolina State University, Department of Botany, BOX 7612, Raleigh, NC 27695, U.S.A. 6 To whom correspondence should be addressed. 751

(Frachisse et al. 1990). On another hand, there are now numerous reports indicating the involvement of calcium after environmental stimulation (Braam and Davis 1990, Haley 1995) and movements of this cation may be one of the first consequences of the plant perception of a stimulation. It is thus not surprising that the slow wave observed after an injurious stimulation is dramatically dependant upon the availability of calcium (Julien et al. 1991): incubating the plants in the presence of the calcium chelator EGTA, or the calcium channel blocker LaCl3 strongly reduce its amplitude. In addition, we have recently demonstrated a strong correlation between membrane potential and the accumulation of the calmodulin transcripts (Vian et al. 1996). However, no investigations have been made to study the accumulation of calmodulin transcripts, used as a cellular marker of stress perception, under experimental conditions known to reduce the amplitude of the SW after an injurious stimulus. The main aim of this research is to determine if the wound signal is transmitted from the wounded cotyledon to the intact, distant hypocotyl, and transduced in a cellular response (i.e. causing a calmodulin mRNA accumulation) when the plants are cultivated in the presence of calcium counteracting drugs. Bidens pilosa L. var radiata (Compositae) was cultivated as described earlier (Vian et al. 1993), treated with chemicals for 24 h and then wounded by pricking the cotyledons. The tissues were harvested at various times after the treatment, and then immediately frozen in liquid nitrogen. RNA was isolated as described by Bogorad et al. (1983), and cDNA library was constructed in the bacteriophage Agt-10 (Stratagene) from 5//g of poly(A)+RNA isolated from 60 min wounded plants, using a commercially available kit (Pharmacia). A calmodulin cDNA clone was obtained from this library using the tch-1 cDNA as a probe (Its sequence is available in the EMBL, Genebank and DDBJ databases under the accession number X89890). Northern blots analyses were performed as described by Sambrook et al. (1989), using the V-3124 (B. pilosa calmodulin cDNA) as a probe.

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Local and distant responses to wounding

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Fig. 1 Accumulation of calmodulin transcripts after wounding. Northern blot showing the time course of CaM transcripts accumulation in the cotyledons (A), or hypocotyls (B) after wounding. The plants, transferred on day 5 to deionized water, were left untreated (lanes 1) or wounded and harvested 5 (lanes 2), 30 (lanes 3) or 60 min (lanes 4) later. CaM: hybridization of total RNA with B.pilosa calmodulin cDNA. 18S: control of RNA loading with 18S ribosomal RNA probe.

We isolated total RNA from cotyledons and hypocotyls of plants transferred 24 h earlier onto deionized water, then wounded and harvested 5, 30 or 60 min later. The blot was probed with V-3124 cDNA (Fig. 1). The steady state level of the corresponding transcripts was low both in the cotyledons (Fig. 1A, lane 1) and in the hypocotyl (Fig. IB, lane 1). The highest level of transcripts was detected 30-60 min after treatment both in the cotyledons (Fig. 1A, lanes 3-4) and in the hypocotyl (Fig. IB, lanes 3 4). At these times the typical increase in CaM mRNA level was 6-7-fold in both the cotyledons and the hypocotyl. Effect of CaCl2—Concentrations of CaCl2 ranging from 1 to 0.01 mM (Fig. 2A, lanes 3-5) did not induce by themselves CaM mRNA accumulation compared to unwounded plants grown on water (Fig. 2A, lane 1). However, at all calcium concentrations, wounding these plants resulted in the accumulation of CaM transcripts (Fig. 2A, lanes 6-8). The accumulation was higher (6-fold) when plants were incubated on 1 mM and 0.1 mM CaCl2 (Fig. 2A, lanes 6-7) than on lower concentrations (4.5 fold, lane 8). Effect of EGTA andLaCl3—Surprisingly, high concentration of EGTA (10 mM) did not totally prevent the CaM transcripts accumulation, which was 2.5 fold in wounded plants (Fig. 2B, lanes 3 and 7). In contrast, concentrations of 0.1 and 1 mM of EGTA (Fig. 2B, lanes 4-5) prevented the accumulation of CaM transcripts after wounding (Fig. 2B lanes 8 and 9). Lower concentrations of EGTA (0.01 mM, Fig. 2B lane 6) did not prevent the CaM mRNA accumulation (Fig. 2B, lane 10). Treatments of the plants with 0.1 mM LaCl3 (calcium channel blocker) prevented the accumulation of CaM transcripts in the hypocotyl (Fig. 2C, lanes 1-2) after wounding the cotyledons, with almost no variation of the CaM tran-

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Fig. 2 (A) Effect of CaCl2 on calmodulin mRNA accumulation. Plants were placed for 24 h on deionized water (lanes 1-2) or on solution containing CaCI2 at 1 mM (lanes 3 and 6), 0.1 mM (lanes 4 and 7) or 0.01 mM (lanes 5 and 8). RNA was isolated from control (lanes 1 and 3-5) or 60 min wounded plants (lanes 2 and 6-8). (B) Effect of EGTA on calmodulin mRNA accumulation. Plants were placed for 24 h on deionized water (lanes 1-2) or on a solution containing EGTA at 10 mM (lanes 3 and 7), 1 mM (lanes 4 and 8), 0.1 mM (lanes 5 and 9) and 0.01 mM (lanes 6 and 10). RNA was isolated from control (lanes 1 and 3-6) or 60 min wounded plants (lanes 2 and 7-10). (C) Effect of the calcium channel blocker LaCl3 on calmodulin mRNA accumulation. Plants were placed for 24 h on a solution containing 0.1 mM LaC^. The plants were left unwounded (lanes 1, 3) or wounded for 60 min (lanes 2, 4). RNA was isolated from hypocotyls (lanes 1, 2) or cotyledons (lanes 3, 4). CaM: hybridization with B. pilosa calmodulin cDNA. 18S: control of RNA loading with 18S ribosomal RNA probe.

script level in the wounded cotyledons (Fig. 2C, lanes 3-4). The first goal of this work was to determine whether calmodulin mRNA accumulation in response to wounding occurred in just the tissue wounded, or in a distant tissue as implied by the work of Braam and Davis (1990), thus implying the transmission of a wound signal. The second goal was to study the involvement of the wound-induced wave of depolarization, which has been demonstrated to be linked to calmodulin mRNA accumulation (Vian et al. 1996).

Local and distant responses to wounding

After cotyledon wounding on plant growth on water, an increase in calmodulin transcripts occurred within 30 min in both the cotyledons themselves and in the distant hypocotyl (Fig. 1). The kinetics of accumulation were essentially the same at the site of the wounding and at a distance suggesting that the transmission/transduction of the wound information from the cotyledon to the hypocotyl occurs very rapidly. The nature of this traumatic signal is complex: previous reports in Solanaceae have demonstrated that oligosaccharides (Ryan and Farmer 1991), systemin (Pearce et al. 1991), abscisic acid (Pefia-Cortes et al. 1991, 1996) and electric depolarization (Stankovic and Davies 1996) are part of the signaling system. With regards to this last aspect, it was shown in B. pilosa (Julien et al. 1991) that incubating plants in the presence of EGTA or LaCl3 caused a massive (almost 60%) reduction in the amplitude of the wound-generated "slow wave". These compounds are widely employed to study calcium metabolism (Moyen and Roblin 1993, Haley et al. 1995) and the concentrations used in the present study, while relatively high, are similar to, or lower than those used for study on whole plants (Moysset et al. 1994, Grill 1995). We showed in this paper that after similar treatments, the CaM mRNA accumulation is highly reduced (Fig. 2B, C) although calcium treatment do not affect the response (Fig. 2A). The results obtained here reinforce the relationship between the membrane potential and CaM gene expression that we demonstrated recently (Vian et al. 1996) and may be an indication for the involvement of membrane potential in signal transmission and gene expression. We wish to thank Dr. Janet Braam (Rice University, Houston, U.S.A.) for the gift of the tch-1 cDNA clone and Dr. Claude Jean (University Blaise Pascal, Clermont-Ferrand, France) for the gift of the 18S ribosomal probe. Thanks to Dana Moxley (NCSU, Raleigh, U.S.A.) for photographic work and to NSF for grant No IBN-93-10508 awarded to E.D. This research has been supported by INRA/Universite Blaise Pascal, and by doctoral fellowships from the Ministere de l'Enseignement Superieur et de la Recherche (FRANCE) awarded to A.V. and C.H.V.

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(Received November 19, 1996; Accepted April 10, 1997)