A genetic approach to study cGMP signaling in plants

The occurrence of the second messenger 3,5-cyclic guanyl monophosphate (cGMP) has been shown in a number of plant species, including barley, tobacco and Arabidopsis. Physiological processes where cGMP signaling has been observed or inferred to play a role include chloroplast development, primary and lateral root growth, α-amylase production in aleurone tissue, stomatal movements, NO-dependent expression of defence-related genes and salt/osmotic stress. Although there have been few reports about putative guanylate cyclases in Arabidopsis thaliana but still to date a genuine NO dependent guanylate cyclase has not been identified in plants. For this reason cGMP levels could not be manipulated by genetical tools and it has been studied by pharmacological approach. In this work we have developed a novel approach for studying cGMP signaling by expressing mammalian soluble guanylate cyclase in plants. Transgenic Arabidopsis thaliana plants have been developed which constitutively express a functional soluble guanylate cyclase (alpha and beta subunits). The expression of both sub-units of the enzyme was confirmed by Real-time PCR. These plants show a considerably higher guanylate cyclase activity and accumulate many folds higher cGMP content as compared to wild type plants. Two lines were selected for further studies; one of the lines accumulating 40-50 fold more cGMP while the other line having 3-4 fold more cGMP than wild type plants. At phenotypic level, both transgenic plants have shorter roots than wild type plants and exhibit a salt stress sensitive phenotype. Further more the line with higher cGMP level was resistant to both virulent and avirulent strains of Pseudomonas syringe. By micrarray technology, cGMP mediated transcriptomic changes were studied in the line with higher cGMP level. cGMP was able to modulate the transcription of 227 genes. The modulated genes belong to diverse physiological processes like transport, metabolism, biotic/ abiotic stress response, transcription and hormone stimulus response. These plants are a powerful tool to study the role of cGMP siganling in plants.