Plant cyclic nucleotides (cGMP and cAMP) have been reported to be involved in several physiological processes, but the mechanisms of action and the following signal transduction events, are still poorly characterized. The main object of the 2010 FIRB project is the study of cyclic nucleotide roles in signalling events following biotic stress in Arabidopsis thaliana. For this aim transgenic plants with altered levels of both cGMP and cAMP, have been generated. Specifically, plants over-expressing a mammalian guanylate cyclase (GC) or a phosphodiesterase (PDE) to alter the cGMP levels, and plants over-expressing a chimeric protein namely "cAMP-sponge" (cAS), to buffer the cAMP, were produced. Moreover, in order to measure both in vivo and in vitro the cNMP levels and dynamics, different approaches have been followed: i) production of transgenic lines expressing genetically encoded FRET-based probes (EPACs and Red-cGES) and ii) set up of alpha screen technology for plant tissue. To gain insight into the inter-relationships of cNMPs and other second messengers in the plant signalling events, we then crossed the plants with altered levels of cNMPs with Arabidopsis transgenic lines expressing the genetically encoded probes Cameleon (for in vivo Ca2+ detection) and roGFP2 (for in vivo detection of redox potential). In Arabidopsis, it has been reported that Ca2+, redox status and cNMPs play important signalling roles in response to the avirulent attack of Pseudomonas syringae. Our preliminary results, using Cameleon and roGFP2 wild type plants, showed a transient increase of both Ca2+ and oxidation status upon avirulent attack of Pseudomonas syringae. Biochemical analyses confirmed that both Glutathione and Ascorbate pools were more oxidized upon such attack. In order to study if and how the cNMPs play a role in the regulation of Ca2+ and redox status similar experiments will be performed with the different generated lines with altered levels of cNMPs. Finally, to understand if the altered levels of cNMPs can affect gene expression in response to the considered pathogen attack, a transcriptomic and proteomic characterization of the transgenic lines will be performed. Following this multidisciplinary approach, the understanding of the specific biological responses regulated by cAMP and cGMP and the downstream signalling events actually represents a big challenge in plants
CYCLIC NUCLEOTIDES IN BIOTIC STRESS SIGNALLING IN PLANT: FIRB PROJECT OUTLINES
SABETTA W;BLANCO E
2014
Abstract
Plant cyclic nucleotides (cGMP and cAMP) have been reported to be involved in several physiological processes, but the mechanisms of action and the following signal transduction events, are still poorly characterized. The main object of the 2010 FIRB project is the study of cyclic nucleotide roles in signalling events following biotic stress in Arabidopsis thaliana. For this aim transgenic plants with altered levels of both cGMP and cAMP, have been generated. Specifically, plants over-expressing a mammalian guanylate cyclase (GC) or a phosphodiesterase (PDE) to alter the cGMP levels, and plants over-expressing a chimeric protein namely "cAMP-sponge" (cAS), to buffer the cAMP, were produced. Moreover, in order to measure both in vivo and in vitro the cNMP levels and dynamics, different approaches have been followed: i) production of transgenic lines expressing genetically encoded FRET-based probes (EPACs and Red-cGES) and ii) set up of alpha screen technology for plant tissue. To gain insight into the inter-relationships of cNMPs and other second messengers in the plant signalling events, we then crossed the plants with altered levels of cNMPs with Arabidopsis transgenic lines expressing the genetically encoded probes Cameleon (for in vivo Ca2+ detection) and roGFP2 (for in vivo detection of redox potential). In Arabidopsis, it has been reported that Ca2+, redox status and cNMPs play important signalling roles in response to the avirulent attack of Pseudomonas syringae. Our preliminary results, using Cameleon and roGFP2 wild type plants, showed a transient increase of both Ca2+ and oxidation status upon avirulent attack of Pseudomonas syringae. Biochemical analyses confirmed that both Glutathione and Ascorbate pools were more oxidized upon such attack. In order to study if and how the cNMPs play a role in the regulation of Ca2+ and redox status similar experiments will be performed with the different generated lines with altered levels of cNMPs. Finally, to understand if the altered levels of cNMPs can affect gene expression in response to the considered pathogen attack, a transcriptomic and proteomic characterization of the transgenic lines will be performed. Following this multidisciplinary approach, the understanding of the specific biological responses regulated by cAMP and cGMP and the downstream signalling events actually represents a big challenge in plantsI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
