Detection and characterization of calcineurin-like activity in Brassica juncea and its activation by low temperature

Here we describe a method for detecting calcineurin-like activity in Brassica juncea seedlings. The activity was standardized with respect to all the assay components. The optimum reaction time for the assay was found to be 10 min at 0.75 μM of R II phosphopeptide, a specific substrate for calcineurin. Stimulation of activity by CaM (0.1 μM) and CaCl2 (1 mM) was observed. The enzyme showed maximum activity in 125 mM Tris, 200 mM NaCl and 20 mM MgCl2 solution. The activity was differentially distributed in root, shoot and hypocotyls. It was maximum in roots (2.8 nM PO4 released/mg protein), followed by hypocotyls (0.95 nM PO4 released/mg protein) and cotyledonary leaves (0.85 nM PO4 released/mg protein), respectively. Low temperature (LT) stress treatment (4 °C) of short durations (5 and 15 min) showed a substantial increase in the activity. Maximum increase was observed in cotyledonary leaves (34.8%), followed by roots (25.6%) and hypocotyls (5.25%), respectively after LT treatment of 5 min suggesting its probable involvement in early signaling events. Besides, in vitro phosphorylation studies also showed activation of phosphatase by LT. Hence, the study indicates probable involvement of calcineurin-like activity in early cold stress signaling. Moreover, this optimized activity assay could be adopted to detect calcineurin-like activity in other plants.