Abstract
BackgroundCyclic AMP (cAMP) and cyclic GMP (cGMP) have roles in relaying external signals and modifying gene expression within cells in all phyla. Currently there are no reporter systems suitable for bacteria and plant cells that measure alterations in downstream gene expression following changes in intracellular levels of cyclic nucleotides. As the plant protein OLIGOPEPTIDE TRANSPORTER X (OPTX) is upregulated by cGMP, we fused the OPTX promoter to a luciferase reporter gene (OPTX:LUC) to develop a plant cell reporter of cGMP-induced gene expression. We prepared a second construct augmented with three mammalian cGMP response elements (OPTXcGMPRE:LUC) and a third construct containing five gibberellic acid response elements (OPTXGARE:LUC). All three constructs were tested in bacteria and isolated plant protoplasts.ResultsMembrane permeable cGMP enhanced luciferase activity of OPTX:LUC and OPTXGARE:LUC in protoplasts. Treatment with the plant hormone gibberellic acid which acts via cGMP also generated downstream luciferase activity. However, membrane permeable cAMP induced similar responses to cGMP in protoplasts. Significantly increased luciferase activity occurred in bacteria transformed with either OPTXcGMPRE:LUC or OPTXGARE:LUC in response to membrane permeable cAMP and cGMP. Bacteria co-transformed with OPTXcGMPRE:LUC or OPTXGARE:LUC and the soluble cytoplasmic domain of phytosulfokine receptor1 (PSKR1; a novel guanylate cyclase) had enhanced luciferase activity following induction of PSKR1 expression.ConclusionsWe have developed promoter reporter systems based on the plant OPTX promoter that can be employed in bacteria and isolated plant cells. We have shown that it can be used in bacteria to screen recombinant proteins for guanylate cyclase activity as increases in intracellular cGMP levels result in altered gene transcription and luciferase activity.
Highlights
Cyclic AMP and cyclic GMP have roles in relaying external signals and modifying gene expression within cells in all phyla
SALT OVERLY SENSITIVE 3 (SOS3), CATION/H+ EXCHANGER 21 (CHX21) and OLIGOPEPTIDE TRANSPORTER X (OPTX) all showed at least a two fold increase in expression that was verified by quantitative RT-PCR [29]
We determined that SOS3, CHX21 and OPTX were all expressed in freshly isolated leaf mesophyll protoplasts (Figure 3A) despite these genes being originally identified in root tissue [29]
Summary
Cyclic AMP (cAMP) and cyclic GMP (cGMP) have roles in relaying external signals and modifying gene expression within cells in all phyla. Promoter reporter constructs containing the cAMP binding protein response element have been extensively used in mammalian systems to detect changes in cAMP that alter gene expression [22,23,24,25]. The complexities of the interaction between the cyclic nucleotide pathways has led to the development of a mammalian cGMP reporter system pathways using the cAMP response element and overexpressed protein kinase G but this system is unable to discriminate between cAMP and cGMP [28] Despite these advances in mammalian cells, a reporter assay for plant cells that detects gene expression induced by changes in intracellular levels of either cGMP or cAMP levels is lacking
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