Key-words: Lycopersicon esculentum; aequorin; calcium; channels; systemin; tomato; wound response. INTRODUCTION Synthesis and accumulation of proteinase inhibitors (PIs)is one of the responses induced in plants by wounding. This response occurs both in the wounded leaf and in distal unwounded leaves within a few hours of the initial damages (Pearce et al. 1991;O'Donnell 1995). This implies the systemic propagation of a signal from the injury site to distal tissues. Putative chemical messengers for this systemic wound signal include systemin. Systemin is an 18 amino acid polypeptide which was originally isolated from tomato (Lycopersicon esculentum) leaves and which, like wounding itself, elicits the systemic synthesis of PIs in tomato plants (Pearce et al. 1991). Systemin was named after its property of being systemically mobile in the phloem (Pearce et al. 1991). Following its application to wound sites on leaves, the polypeptide moves throughout the leaf and is found within 1-2 h in the phloem exudate (Pearce et al. 1991;Narvaez-Vasquez et al. 1995). Systemin, the first plant polypeptide hormone, behaves in a strikingly similar manner to polypeptide hormones in animal and yeast cells . However, until now the initial stages in stimulus-response coupling at the plasma membrane have not been clear. The signal transduction pathway is thought to involve the binding of systemin to a membrane receptor (Farmer & Ryan 1992) which has not yet been clearly identified. Schaller & Ryan (1994) found a 50 kDa plasma membrane protein which binds systemin. However, this proteolytic protein does not appear to be a receptor, but has the properties of a furinlike proteinase able to cleave systemin to smaller polypeptides (Schaller & Ryan 1994).The binding of systemin to a plasma membrane receptor would lead to the activation of a lipase and then to a subsequent intracellular release of linolenic acid and synthesis of methyl jasmonate (Farmer & Ryan 1990. These two components of the octadecanoid signal transduction pathway are known to have a powerful ability to induce Pin II gene expression (Farmer & Ryan 1990. Systemin and jasmonic acid (JA) have also now been shown to trigger other cell responses such as synthesis of woundinducible polyphenol oxidase (Constabel, Bergey & Ryan 1995) and aspartic protease [Schaller & Ryan 1996; see also ]. Moyen & Johannes (1996) recently showed that some of the early events triggered by systemin within a few minutes of application on Lycopersicon esculentum mesophyll tissue are a depolarization of the plasma membrane and a transient H + efflux followed by a longer lasting H + influx. Moreover, systemin also triggers an increase of extracellular K + par- 1101alleled by an alkalization in the extracellular medium of suspension-cultured cells of Lycopersicon peruvianum (Felix & Boller 1995). Systemin-induced ion fluxes are affected in the presence of fusicoccin, which has been shown to antagonize the PI synthesis induced by systemin (O'Donnell 1995) and by oligogalacturonides (OG) (Doherty & Bowle...

Protoplasts isolated from root cap cells of maize were shown to secrete fucose-rich polysaccharides and were used in a patch-clamp study to monitor changes in whole-cell capacitance. Ca2+ was required for exocytosis, which was measured as an increase in cell capacitance during intracellular dialysis with Ca2+ buffers via the patch pipette. Exocytosis was stimulated significantly by small increases above normal resting [Ca2+]. In the absence of Ca2+, protoplasts decreased in size. In situ hybridization showed significant expression of the maize annexin p35 in root cap cells, differ-entiating vascular tissue, and elongating cells. Dialysis of protoplasts with maize annexins stimulated exocytosis at physiological [Ca2+], and this could be blocked by dialysis with antibodies specific to maize annexins. Dialysis with milli-molar concentrations of GTP strongly inhibited exocytosis, causing protoplasts to decrease in size. GTPgammaS and GDPbetaS both caused only a slight inhibition of exocytosis at physiological Ca2+. Protoplasts were shown to internalize plasma membrane actively. The results are discussed in relation to the regulation of exocytosis in what is usually considered to be a constitutively secreting system; they provide direct evidence for a role of annexins in exocytosis in plant cells.

Protoplasts isolated from root cap cells of maize were shown to secrete fucose-rich polysaccharides and were used in a patch-clamp study to monitor changes in whole-cell capacitance. Ca2+ was required for exocytosis, which was measured as an increase in cell capacitance during intracellular dialysis with Ca2+ buffers via the patch pipette. Exocytosis was stimulated significantly by small increases above normal resting [Ca2+]. In the absence of Ca2+, protoplasts decreased in size. In situ hybridization showed significant expression of the maize annexin p35 in root cap cells, differ-entiating vascular tissue, and elongating cells. Dialysis of protoplasts with maize annexins stimulated exocytosis at physiological [Ca2+], and this could be blocked by dialysis with antibodies specific to maize annexins. Dialysis with milli-molar concentrations of GTP strongly inhibited exocytosis, causing protoplasts to decrease in size. GTPgammaS and GDPbetaS both caused only a slight inhibition of exocytosis at physiological Ca2+. Protoplasts were shown to internalize plasma membrane actively. The results are discussed in relation to the regulation of exocytosis in what is usually considered to be a constitutively secreting system; they provide direct evidence for a role of annexins in exocytosis in plant cells.

The plant polypeptide signal systemin induces proteinase inhibitor synthesis in tomato leaves. We show here that systemin elicits a transient depolarization of the tomato mesophyll cell membrane. Furthermore it triggers a transient decrease in the external pH of the mesophyll tissue which is followed by a sustained pH increase. In the presence of fusicoccin (which has been shown to antagonize the synthesis of proteinase inhibitors) the depolarization and transient H+ efflux are attenuated whereas the slower phase of the sustained electroneutral H+ influx persists. These results suggest that systemin‐induced changes in ion transport play a role in the early phases of systemin signal transduction.

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