In previous studies of the calcicoles Centaurea scabiosa and Leontodon hispidus, the stomata on isolated epidermis closed partially when the concentration of calcium in the medium was above 1 mol m"'^. This is a much smaller concentration than that helieved to be delivered into the leaves in xylem sap when the plants are growing in a calcium-rich medium, and hence the mechanism for 'protecting' stomata from excessive exposure to free calcium is thought to be of great physiological significance. It is shown here that, in the leaves of both species, a substantial amount of the calcium they contain is located within mesophyll cells, and virtually all of that which does enter the epidermis is contained within trichomes, probably as calcium oxalate. The amounts of calcium in the vicinity of the stomata thus remain small despite high concentrations elsewhere, ensuring that the essential role of Ca^* in intracellular signalling in guard cells can continue to be performed without disturbance. MATERIALS AND METHODS Plant culturePlants of Centaurea scahiosa L, (greater knapweed) and Leontodon hispidus L, (rough hawkbit) were raised from 880

Regulation of the concentration of Ca2+ in the apoplast of plants is essential in order to allow Ca(2+)-dependent processes, such as Ca(2+)-mediated signal transduction in stomatal guard cells, to function correctly. This is particularly important for plants growing with high levels of Ca2+ in the rhizosphere. Recently, we have shown that in two calcicoles, Leontodon hispidus L. and Centaurea scabiosa L., trichomes play a key role in this regulatory process. Ozone is known to have a marked effect on plant Ca2+ homeostasis. Therefore, we have examined the effect of this pollutant on the regulation by trichomes of apoplastic Ca2+ in the calcicole L. hispidus. Treatment with 100 nl l-1 ozone resulted in a reduction in stomatal conductance of approximately 25% in plants grown with 15 mM Ca2+ in the rhizosphere. Analysis of total Ca2+ levels revealed that these changes in stomatal behaviour reflect a decrease in the ability of trichomes to sequester Ca2+. The amount of Ca2+ present in the trichome tip cell was reduced by approximately 38%. This was accompanied by an increase in the levels of Ca2+ in the guard cells and other tissues of the leaf. These data suggest that ozone has a detrimental effect on the ability of trichomes to regulate the concentration of apoplastic Ca2+ in L. hispidus, resulting in altered stomatal behaviour, and hence gaseous exchange, possibly due to the disruption of guard-cell Ca(2+)-mediated signal transduction. This has important implications for the growth and survival of plants growing in Ca(2+)-rich soils.