Transgenic aequorin monitors cytosolic calcium transients in soybean cells challenged with β-glucan or chitin elicitors

Abstract

Transgenic soybean (Glycine max L.) cells expressing aequorin were used to monitor changes in cytosolic Ca2+ concentrations in response to treatment with fungal elicitors. After an apparent lag phase of about 60 s, both chitin fragments and β-glucan elicitors caused a rapid increase in cytosolic Ca2+ concentration, which peaked within 2–2.5 min of treatment. The Ca2+ concentration then decreased and reached the basal level after about 5 min in the case of the treatment with chitin fragments, while a second rise in the Ca2+ concentration with a maximum occurring after about 7–8 min was observed in the case of β-glucan treatment. Calibration of the signals showed that the elicitors enhanced the cytosolic Ca2+ concentration from resting concentrations as low as 0.1 lM to highest levels of about 2 lM. Dose-response experiments showed that the concentration of elicitors giving a Ca2+ response at the 50% level was 0.4 nM for the chitin fragment and 28 lM and 72 lM, respectively, for a synthetic hepta-β-glucoside and a fungal β-glucan fraction. The β-glucan- or N,N′,N′′,N′′′-tetraacetyl chitotetratose (CH4)-induced Ca2+ signals were inhibited by both the Ca2+ chelator 1,2-bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and by the Ca2+-channel inhibitor La3+. Neomycin, whose target in plant cells has not yet been clearly identified, reduced predominantly the expression of the second peak of the biphasic Ca2+ curve following β-glucan treatment. Bacterial cyclic β-glucans known to suppress β-glucan-induced phytoalexin production were also found to function as a suppressor for the Ca2+ response that was elicited by the fungal β-glucans. The results clearly show that the increase in the cytosolic Ca2+ concentration is an early and rapid event in the elicitor-sensing mechanism of soybean cells, and is probably connected with the subsequent activation of defence responses.