Calcium and calcium-dependent protein kinases are involved in nitric oxide- and auxin-induced adventitious root formation in cucumber

Abstract

A few years ago it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during adventitious root (AR) formation in cucumber (Cucumis sativus). More recently, a mitogen-activated protein kinase cascade was shown to be induced by IAA in a NO-dependent, but cGMP-independent, pathway. In the present study, the involvement of Ca²⁺ and the regulation of Ca²⁺-dependent protein kinase (CDPK) activity during IAA- and NO-induced AR formation was evaluated in cucumber explants. The effectiveness of several broad-spectrum Ca²⁺ channel inhibitors and Ca²⁺ chelators in affecting AR formation induced by IAA or NO was also examined. Results indicate that the explants response to IAA and NO depends on the availability of both intracellular and extracellular Ca²⁺ pools. Protein extracts from cucumber hypocotyls were assayed for CDPK activity by using histone IIIS or syntide 2 as substrates for in-gel or in vitro assays, respectively. The activity of a 50 kDa CDPK was detected after 1 d of either NO or IAA treatments and it extended up to the third day of treatment. This CDPK activity was affected in both extracts from NO- and IAA-treated explants in the presence of the specific NO-scavenger cPTIO, suggesting that NO is required for its maximal and sustained activity. The in-gel and the in vitro CDPK activity, as well as the NO- or IAA-induced AR formation, were inhibited by calmodulin antagonists. Furthermore, the induction of CDPK activity by NO and IAA was shown to be reliant on the activity of the enzyme guanylate cyclase.