Abstract
Single-celled spores of the fern Ceratopteris richardii undergo gravity-directed cell polarity development that is driven by polar calcium currents. Here we present results that establish a role for nitric oxide (NO)/cGMP signaling in transducing the stimulus of gravity to directed polarization of the spores. Application of specific NO donors and scavengers inhibited the calcium-dependent gravity response in a dose-dependent manner. The effects of NO donor exposure were antagonized by application of NO scavenger compounds. Similarly, the guanylate cyclase inhibitors 6-anilino-5,8-quinolinedione and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin, and the phosphodiesterase inhibitor Viagra, which modulate NO-dependent cGMP levels in the cells, disrupted gravity-directed cell polarity in a dose-dependent manner. Viagra effects were antagonized by application of NO scavengers, consistent with the postulate that NO and cGMP are linked in the signaling pathway. To identify other components of the signaling system we analyzed gene expression changes induced by Viagra treatment using microarrays and quantitative real-time reverse transcription-polymerase chain reaction. Preliminary microarray analysis revealed several genes whose expression was significantly altered by Viagra treatment. Three of these genes had strong sequence similarity to key signal transduction or stress response genes and quantitative real-time reverse transcription-polymerase chain reaction was used to more rigorously quantify the effects of Viagra on their expression in spores and to test how closely these effects could be mimicked by treatment with dibutyryl cGMP. Taken together our results implicate NO and cGMP as downstream effectors that help link the gravity stimulus to polarized growth in C. richardii spores. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers BE 640669 to BE 643506, BQ 086920 to BQ 087668, and CV 734654 to CV 736151.
Highlights
Single-celled spores of the fern Ceratopteris richardii undergo gravity-directed cell polarity development that is driven by polar calcium currents
While the SDs do reflect the variations within the population these values are not normalized because they are based on the average values for each treatment
Our experiments clearly show that treatment of the spores with nitric oxide (NO) donors or scavengers disrupts gravity-dependent polarity in the fern spore system
Summary
Single-celled spores of the fern Ceratopteris richardii undergo gravity-directed cell polarity development that is driven by polar calcium currents. Preliminary microarray analysis revealed several genes whose expression was significantly altered by Viagra treatment Three of these genes had strong sequence similarity to key signal transduction or stress response genes and quantitative real-time reverse transcription-polymerase chain reaction was used to more rigorously quantify the effects of Viagra on their expression in spores and to test how closely these effects could be mimicked by treatment with dibutyryl cGMP. In animals NOS has been verified to be the enzyme that initiates the NO signaling cascade and a plant enzyme associated with NOS activity, termed AtNOS1 (Guo et al, 2003), has recently been described. The Arabidopsis (Arabidopsis thaliana) At-NOS1 activity is independent of tetrahydrobioprotein and flavin, but is regulated by Ca21-calmodulin and is NADPH dependent (Guo et al, 2003)
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