It was revealed that various injured or infected organs

It was revealed that various injured or infected organs of Hippeastrum produce a mixture of an orange-colored chalcone and flavans which can be oxidized to red-colored dimer (Wilmowicz et al., 2014, Wink and Lehmann, 1996), preventing the penetration of injured tissues by Phoma narcissi, Botrytis cinerea and Fusarium oxysporum (Saniewska et al., 2005). The biosynthesis pathways of these phytoalexin-like compounds in wounded tissues of Hippeastrum are unknown, but efficient induction of immune processes is known to depend on different internal signaling pathways, among which cyclic nucleotides play an important role. Consequently, we made an effort to identify the guanylyl cyclase gene and develop an efficient method for plant guanylyl cyclase protein production. Moreover, the GC neurokinin receptor antagonist profile and the endogenous content of cGMP in Hippeastrum scales, mechanically wounded or wounded and then infected by fungus Peyronellaea curtissi (=Phoma narcissi), were investigated to confirm the participation of the cGMP signaling pathway in post-wounding reactions.
Materials and methods
Results and discussion The common occurrence of cGMP in plants may illustrate its importance. The cellular level of cGMP is controlled by cyclases and phosphodiesterases. In this paper we wished to address the following questions: is guanylyl cyclase present in Hippeastrum plant? If so, is it involved in particular stress conditions (wounding and infection)? To answer these questions the putative GC coding gene was characterized, recombinant guanylyl cyclase was produced, HpGC1 expression pattern was analyzed, and finally, the endogenous level of cGMP was measured in two stress regimes.
Acknowledgements The authors thank Jon Callaghan for English correction and Grażyna Czeszewska-Rosiak (Nicolaus Copernicus University, Toruń, Poland) for technical assistance. This project was supported by the National Science Centre, grants No. NN310141435 and NN310 301839 and funds provided by Nicolaus Copernicus University (Toruń, Poland) for the research program of the Chair of Plant Physiology and Biotechnology.
Introduction C-type natriuretic peptide (CNP) is a paracrine factor that stimulates the growth of long bones and vertebrae, promotes axonal bifurcation in the spinal cord, and prevents resumption of meiosis in the ovarian follicle [1], [2], [3]. These physiologic functions of CNP are mediated by guanylyl cyclase-B (GC-B), which elevates intracellular cGMP in response to CNP binding. Female mice lacking GC-B are infertile, and mice of both sexes lacking functional CNP or functional GC-B exhibit disproportionate dwarfism caused by reduced chondrocyte proliferation and hypertrophy [4], [5]. In humans, genetic mutations that inactivate both alleles encoding GC-B cause acromesomelic dysplasia, type Maroteaux (AMDM) dwarfism [6]. Conversely, mutations that increase CNP expression [7], [8] or mutations that activate a single GC-B allele in the absence of CNP cause skeletal overgrowth [9], [10], [11]. CNP levels in plasma are also predictive of longitudinal bone growth [12], [13]. GC-B is a single membrane-spanning enzyme that exists as a higher ordered oligomer, possibly a dimer, that catalyzes the synthesis of cGMP from GTP in response to CNP binding [14]. The extracellular domain of GC-B is glycosylated and terminal N-linked glycosylation is required for the formation of an active GC catalytic domain [15], [16]. AMDM dwarfism-causing missense mutations are most often associated with receptors lacking terminal N-linked glycosylation that markedly reduces or abolishes the ability of GC-B to form an active catalytic domain [16]. The intracellular portion of GC-B consists of a kinase homology domain that contains six chemically identified serine/threonine phosphorylation sites and one putative, functionally identified serine phosphorylation site [17], [18], [19], a short coiled-coiled dimerization region, and a carboxyl-terminal GC domain [14], [20].