One of the goals of Aim 1 of the original proposal was to molecularly identify cation transporters that could play a major role in driving the trans-cell calcium current that is directed by gravity in fern spores cells. In accord with this goal, we obtained the full-length sequence of a putative MscS (mechanosensitive channel, small current) gene, and obtained evidence in support of the conclusion that it actually has Msc activity by showing that it could partially rescue a bacterial knockout of Msc.  We also obtained partial sequences of two other Msc candidates in Ceratopteris that will serve as the basis to design primers that can “walk” through the Ceratopteris transcriptome and genome to obtain their full sequences, and the primary sequences of the proteins they encode. This will enable the production of antibodies that can be used to identify where each of the Msc channels is localized. If any of these candidates prove to be mechanosensitive channels on the plasma membrane, it (or they) would be the one(s) most likely to undergo post-translational modifications by gravity stimulation.

The goal of Aim 2 of the original proposal was to test the role of genes encoding Ca2+-binding proteins that are expressed at the peak of the trans-cell Ca2+ current by suppressing or overexpressing their transcription using transformation methods. We have successfully generated transgenic Ceratopteris spores expressing one of several different transgenes, and these spores will be valuable tools for genetically dissecting gravity responses in spores that respond to gravity while they are still single cells. The mutants include one expressing the calcium reporter Yellow Cameleon, which will enable assessing gravity-induced changes in [Ca2+]cyt; one constitutively expressing a spore annexin, which is a calcium-binding protein implicated in regulating calcium channels in plasma membranes, one suppressed in annexin expression; one knocked down in expression of the Ca2+ATPase pump that helps drive the gravity-induced trans-cell calcium current; and one knocked down in one of the mechanosensitive ion channels described above in the paragraph on Aim 1. Generating these mutants and genetically confirming their mutant status required over two years of ground-breaking research, and the transgenic products are the first (and still the only) fern mutants modified in spore-expressed genes.

We made the most progress on Aim 3 of the original proposal, which was to test the role of eATP in promoting the critical Ca2+-uptake step of the spore gravity response. Key background information that provides the rationale for this goal is that when gravity directs the polarization of Ceratopteris fern spores this process begins with the uptake of calcium through channels at the bottom of the spore, and this step is necessary for the gravity response. Data from several different laboratories showing that extracellular ATP (eATP) regulates calcium channels in plants and animals led to the hypothesis that extracellular nucleotides could play a role in the gravity-directed polarization of Ceratopteris spores. In animal and plant cells ATP can be released from mechanosensitive channels.

The idea that a differential distribution of a signaling molecule could lead to morphological polarity is not unique. In this regard, gradients of the growth hormone auxin are among the best documented to be inductive of cell polarity in plants. The differential distribution of eATP during gravity-directed polarization could be an early step in a series of asymmetrical molecular events that ultimately lead to the downstream cellular and morphological changes needed to generate a gravity-directed, downward growing rhizoid in Ceratopteris spores. Precedent data relevant to this hypothesis are those of Tang et al. (Wenqiang Tang, Shari R. Brady, Yu Sun, Gloria K. Muday, and Stanley J. Roux. Extracellular ATP Inhibits Root Gravitropism at Concentrations That Inhibit Polar Auxin Transport. Plant Physiol. 2003 131: 147-154.) in the journal Plant Physiology, who showed that a high [eATP] inhibits the gravity response in Arabidopsis roots. They interpreted their data as evidence that an eATP gradient could be one of the first steps involved in the gravity response in plant cells. These data led us to test whether gravity could induce a gradient of eATP between the top and bottom of germinating fern spores, and, if so, whether this gradient could help mediate the gravity effect of directing the polarization of the spores.

In order to carry out this test, an ATP biosensor, developed by the McLamore laboratory at the University of Florida, was used to measure the [eATP] at the bottom and top of germinating spores during gravity-directed polarization. This biosensor could quantify eATP levels down to the low nM range, and it has a tip diameter of only 1-2 µm, much smaller than the 100-120 µm diameter of spore cells. Using this unique tool with a micromanipulator that could guide the probe to within a few microns of the spore surface, we made the exciting discovery that the [eATP] along the bottom of the spore averaged 7-fold higher than the concentration at the top. Treatments that disrupted the gradient, such as flooding the medium with ATP, or that antagonized eATP receptors, such as adding the ATP receptor blocker PPADS to the medium, or that hydrolyzed ATP in the medium, such as by the addition of phosphatase enzymes, all resulted in a statistically significant decrease in the gravity response. In order to investigate the source of ATP release, spores were treated with Brefeldin A (BFA), which would block cellular secretory activity, and gadolinium trichloride (GdCl3), which would block the transport activity of the mechanosensitive channels that release ATP into the extracellular medium. These treatments resulted in a significant decrease in gravity-directed polarization.

The same eATP biosensor was also used to measure ATP release after treatment with both BFA and GdCl3. Both of these treatments caused a significant decrease in [ATP] measured around spores. These results supported the hypothesis that ATP could be released from mechanosensitive channels and secretory vesicles during gravity-directed polarization.

The novel findings described above show a clear role for eATP in gravity-directed polarization of Ceratopteris spores by documenting that gravity can induce a bottom-to-top gradient of extracellular ATP around the spores, and that this gradient promotes gravity-directed polarization of the spores. They provide support for the hypothesis that mechanosensitive channels, which have been implicated in other reports as critical components of gravity responses, can help generate eATP gradients that can enhance a cellular gravity response. Overall, they reveal that quantifying and evaluating gradients of eATP can lead to a more thorough understanding of how plants use ATP release to direct growth and development.

Production of the novel self-referencing biosensor used to document the gravity-induced eATP gradient between the top and bottom of vertically-oriented spore cells would not have been possible without support from this NASA grant. Recognizing that this biosensor would be a valuable research tool for investigating other eATP-regulated phenomena not only in ferns but also in higher plants, we used it to assay [eATP] levels around growth zones of primary roots of Arabidopsis, because previous research had indicated cells release ATP as they grow. We also used the probe to evaluate whether changing the expression of two apyrase (NTPDase) enzymes (AtAPY1 and AtAPY2), which had been implicated in hydrolyzing and thus limiting the [eATP], actually altered the [eATP]. Our results, which we presented in posters at two international meetings, showed that the [eATP] is highest in root zones that have the highest growth rate (i.e., elongation zones) and that the constitutive expression of AtAPY1 or AtAPY2 lowered this concentration, whereas suppression of these apyrases raised the [eATP]. These results provided novel and important verification of previously postulated hypotheses about the relationship of eATP and apyrase enzymes to growth, and they are being assembled into a manuscript that we expect to submit this summer.

Principal Investigator – Stanley Roux, NNX13AM54G

Year three update on progress of specific aims

Aim 1. Use proteomic, immunological and yeast-based activity assays to determine whether cation transporters that are known to be expressed at the peak of the trans-cell Ca2+ current are localized on the plasma membrane and are regulated post-translationally.

Progress: We have obtained full-length sequence of several putative mechanosensitive ion channels (Msc) from Ceratopteris richardii transcriptome data. Only one of these sequences (tentatively called CrMscC) is expressed in spores throughout germination, including the period of gravity fixation. Bacteria that have mutation in all three of the bacterial Msc genes cannot survive osmotic shock. Exogenous expression of the CrMscC gene in these triple knockout bacteria provides partial rescue to osmotic shock, suggesting the functionality of CrMscC as a mechanosensitive channel on the plasma membrane of bacteria. A collaborator is currently evaluating the mechanosensitive channel activity of the protein through patch clamp analysis in bacteria. We have generated Ceratopteris plants which overexpress this mechanosensitive channel, and plants that have reduced expression of this gene. The expression level of CrMscC is being evaluated in mutant plants and T1 generation of mutants will be obtained. Aim 2. Test the role of Ca2+-binding proteins expressed at the peak of the trans-cell Ca2+ current by suppressing their expression using transformation methods.

Progress: Annexins are calcium-binding proteins, and we have now identified four annexin gene family members in Ceratopteris. One of the roles postulated for annexins in plants is enhancement of calcium channel activity. We are taking a genetic approach to assess the role of annexins in gravity-induced opening of calcium channels in Ceratopteris spores. The transcript expression profile of each of these gene family members has been evaluated in various developmental stages and tissue types. Only one of these genes, AnnCr2, is expressed throughout spore germination. We have exogenously expressed the AnnCr2 protein in E. coli and are using this protein to verify calcium dependent lipid binding of the protein. We have generated Ceratopteris plants that overexpress AnnCr2, and plants that have reduced expression of this gene. The expression level of AnnCr2 is being evaluated in mutant plants, and T1 generation of mutants will be obtained.

We have attempted three times to generate plants that overexpress the plasma membrane calcium ATPase pump (CrACA1), which is expressed strongly at the peak of the trans-cell Ca2+ current. This mutation appears to be lethal to Ceratopteris plants. We have obtained plants with possible suppression of this gene and are evaluating these plants for CrACA1 transcript suppression.

Aim 3. Test the role of eATP in promoting the critical Ca2+-uptake step of the spore gravity response, using extracellular luciferase reporters to assay whether the gravity stimulus induces a bottom-to-top gradient of eATP, and by intracellular reporters to assay changes in [Ca2+]cyt.

Progress: In collaboration with Dr. Eric McLamore at the University of Florida, we have used ATP-selective microelectrodes to confirm that gravity induces an asymmetric gradient of eATP across the Ceratopteris spore before its first cell division, with at least an average of 7X higher [eATP] along the bottom of the spore. Further, we have shown that if this gradient is disrupted by eATP hydrolysis, by flooding the media with extracellular nucleotides, or if the spore is prevented from sensing the gradient using an eATP receptor antagonist, gravity-directed cell polarization is disrupted. Ashley Cannon (grad student) reported on this work in a mini-symposium talk at the American Society for Gravitational and Space Research (ASGSR) meeting in Alexandria, VA last fall. She will also present a poster summarizing this project at the Plant Molecular Biology Gordon Conference and at the American Society of Plant Biologists (ASPB) conference this summer. The manuscript describing these results will be submitted this summer. Spores from plants expressing the FRET-based Ca2+ reporter are currently being screened using Western Blot analysis and fluorescence microscopy. In collaboration with Dr. Ben Smith at The University of Oklahoma, Fluorescence-Lifetime Imaging Microscopy along with a Multiphoton Fluorescence Microscope are being used to visualize intracellular Ca2+ during gravity-directed polarization of Ceratopteris spores.

Other Accomplishments:

We documented that elimination of calcium from spore germination media by chelation during only the first 24 hours of spore development results in reduction of normal gravity directed development, supporting the role of calcium signaling during gravity perception.

We discovered that pharmacological agents that inhibit Golgi vesicle trafficking to the plasma membrane during only the first 24 hours of spore development result in reduction of normal gravity directed development. This suggests a role for protein targeting to the plasma membrane during gravity perception.

We discovered that apyrases and extracellular nucleotides modulate root skewing in Arabidopsis. When plant primary roots grow along a tilted surface that is impenetrable, they can undergo a slanted deviation from the direction of gravity called skewing. Skewing is induced by touch stimuli the roots experience as they grow along the surface. Touch stimuli also induce the release of extracellular ATP (eATP) into plants’ extracellular matrix, and two apyrases (NTPDases) in Arabidopsis, APY1 and APY2, can help regulate the concentration of eATP. The primary roots of seedlings overexpressing APY1 show less skewing than wild-type plants. Plants suppressed in their expression of APY1 show more skewing than wild-type plants. Correspondingly, chemical inhibition of apyrase activity increased skewing in mutants and wild-type roots. Exogenous application of ATP or ATP?S also increased skewing in wild-type roots, which could be blocked by co-incubation with a purinergic receptor antagonist. These results suggest a model in which gradients of eATP set up by differential touch stimuli along roots help direct skewing in roots growing along an impenetrable surface.

We also published the three manuscripts cited below in peer-reviewed journals that all acknowledged support from NASA grant NNX13AM54.

Vanegas DC, Clark G, Cannon AE, Roux S, Chaturvedi P, McLamore ES. 2015. A self-referencing biosensor for real-time monitoring of physiological ATP transport in plant systems. Biosens Bioelectron. 74: 37-44.

Wang X, Ma X, Wang H, Li B, Clark G, Guo Y, Roux S, Sun D, Tang W. 2015. Proteomic study of microsomal proteins reveals a key role for Arabidopsis Annexin 1 in mediating heat stress-induced increase in intracellular calcium levels. Molecular & Cellular Proteomics 14: 686-694

Yang X, Wang B, Farris B, Clark G, Roux SJ. 2015. Modulation of root skewing in Arabidopsis by apyrases and extracellular ATP. Plant & Cell Physiol. 56: 2197-2206.

Abstracts of meeting presentations:

1) A Gradient of Extracellular Nucleotides Directs the Gravity Response in Ceratopteris Spores. Ashley E. Cannon, Diana C. Vanegas, Eric McLamore, Greg Clark, Stanley J. Roux.

2) Gravity-induced calcium ion response of C. richardii during rotation measured by the SporeSat BioCD system. Jenna Rickus, Joonyeong Park, Stanley Roux , Mari Salmi, Antonio Ricco, Daniel Kozarsky, Brittany Wickizer, Abraham Rademacher, Aaron Schooley, Joshua Benton, Adam Sweet , Huyen Tran.

3) Characterization of mechanosensitive channels from a gravi-responsive fern spore. Mari Salmi, Hannah Malcolm, Araceli Cantero-Garcia, Greg Clark, Stanley Roux.

4) Gravity-Induces an Asymmetrical Accumulation of Extracellular Nucleotides that can alter Polarization in Ceratopteris Spores. Ashley E. Cannon, Diana Vanegas, Eric McLamore, Greg Clark, Stanley J. Roux.

Aim 1. Use proteomic, immunological, and yeast-based activity assays to determine whether cation transporters that are known to be expressed at the peak of the trans-cell Ca2+ current are localized on the plasma membrane and are regulated post-translationally.

Progress: Prior work has identified a specific calcium pump, CrACA1, as playing a major role in driving the trans-cell calcium current that is directed by gravity in fern spores cells (Bushart et al., 2013; Bushart et al., 2014). We have hypothesized that a second critical transporter for the trans-cell calcium current would be a plasma membrane localized mechanosensitive cation transporter. As a prerequisite to developing molecular tools to test this hypothesis, we have obtained the full-length sequence of a putative MscS (mechanosensitive channel, small current) gene, and we are now investigating whether it actually has Msc activity by testing whether it can rescue a bacterial knockout of Msc. We also have partial sequences of two other Msc candidates in Ceratopteris and are trying to obtain full-length sequences of them by sequencing BAC-clone DNA. If any of these candidates prove to be mechanosensitive channels we will test whether the protein it encodes is modified post-translationally by gravity stimulation.

Dr. Mari Salmi, the research scientist working on this project, completed the 2014 workshop on Plant Quantitative Proteomics at the University of Wisconsin in July 2014. This workshop gave her training in mass spectrometric techniques, post-translational modification identification, and global proteome quantification. The knowledge and skills gained at this workshop will be applied to evaluation of the proteins involved in regulating the trans-cellular calcium current.

Aim 2. Test the role of Ca2+-binding proteins expressed at the peak of the trans-cell Ca2+ current by suppressing their expression using transformation methods.

Progress: We have successfully generated transgenic Ceratopteris expressing the Calcium reporter Yellow Cameleon, and, using the same transformation methods, we are mid-way toward obtaining other transgenic Ceratopteris sporophytes that are constitutively expressing either a CaATPase or a mechanosensitive channel protein. We believe the channel and the Ca2+ATPase pump we characterized last year are likely to play a major role in generating the trans-cell calcium current that is directed by gravity. Of course we will be using mutant spores modified in their expression of these transporters to clarify how calcium transport mediates the gravity response in germinating spore cells.

Aim 3. Test the role of eATP in promoting the critical Ca2+-uptake step of the spore gravity response, using extracellular luciferase reporters to assay whether the gravity stimulus induces a bottom-to-top gradient of eATP, and by intracellular reporters to assay changes in [Ca2+]cyt.

Progress: In collaboration with Eric McLamore at the University of Florida, we have used ATP-selective microelectrodes to confirm that gravity induces an asymmetric gradient of eATP, with 6X higher [eATP] along the bottom of the spore. Further, we have shown that if this gradient is disrupted by eATP hydrolysis or by flooding the media with extracellular nucleotides, gravity-directed cell polarization is disrupted. Ashley Cannon (grad student) reported on this work in a mini-symposium talk at the American Society of Plant Biologists (ASPB) meeting in Minneapolis this summer. The manuscript describing the construction of the ATP-selective microelectrode and our use of it to measure the [eATP] of roots and spores is now published.

Bushart TJ, Cannon A, Clark G, Roux SJ (2014) Structure and function of CrACA1, the major PM-type Ca2+-ATPase, expressed at the peak of the gravity-directed trans-cell calcium current in spores of the fern Ceratopteris richardii. Plant Biology 16: 151-157.

Bushart TJ, Cannon AE, ul Haque A, San Miguel P, Mostajeran K, Clark GB, Porterfield DM, Roux SJ (2013) RNA-seq analysis identifies potential modulators of gravity response in spores of Ceratopteris (Parkeriaceae): Evidence for modulation by calcium pumps and apyrase activity. American Journal of Botany 100: 161-174.

Aim 1. Use proteomic, immunological, and yeast-based activity assays to determine whether cation transporters that are known to be expressed at the peak of the trans-cell Ca2+ current are localized on the plasma membrane and are regulated post-translationally.

Progress: Prior work has identified a specific calcium pump, CrACA1, as playing a major role in driving the trans-cell calcium current that is directed by gravity in fern spores cells. During this first year of the grant we have further characterizied antibodies to this calcium pump by using them in immunoprecipitation experiments to rapidly separate the pump from other proteins in extracts of fern spores. In these experiments the non-ionic detergent Brij-35 is used to solubilize membranes and allows immunoprecitation of integral membrane proteins as well as cytosolic proteins. We found that the immune serum recognizes at least two bands that are absent in experiments in which pre-immune serum is used. Additionally these same products retain reactivity with the CrACA1 antibody as demonstrated by western blot analysis of immunoprecipitation products. Biochemical identification of these bands is underway. Once recognition of the CrACA1 protein by the antibody has been verified, the antibody will be used to obtain sufficient quantity of the protein for evaluation of post-translational modifications to various stages in the course of gravity response. To do these experiments, novel methods were developed for protein isolation from spores that are in a fixed orientation in agar growth media.

Dr. Mari Salmi, the research scientist working on this project, has been selected to attend the 2014 workshop on Plant Quantitative Proteomics at the University of Wisconsin in July 2014. She will get hands on training in mass spectrometric techniques, post-translational modification identification, and global proteome quantification. The knowledge and skills gained at this workshop will be applied to evaluation of the proteins involved in regulating the trans-cellular calcium current.

Aim 2. Test the role of Ca2+-binding proteins expressed at the peak of the trans-cell Ca2+ current by suppressing their expression using transformation methods.

Progress: Late during this first year of the grant a new, promising method for genetically transforming Ceratopteris spores was published. In preparation for using this new method we are currently creating new plasmid constructs that will allow us to stably suppress two specific genes in Ceratopteris that encode proteins implicated as important for the trans-cell Ca2_ current, annexin and CrACA1.

Aim 3. Test the role of eATP in promoting the critical Ca2+-uptake step of the spore gravity response, using extracellular luciferase reporters to assay whether the gravity stimulus induces a top-to-bottom gradient of eATP, and by intracellular reporters to assay changes in [Ca2+]cyt.

Progress: As described in our proposal we have established a collaboration with Dr. Eric McLamore at The University of Florida to use an ATP-specific microelectrode, to measure the [eATP] on the top and bottom of spores. In preliminary trials, the [eATP] at the bottom of the spore is over two-times higher then the concentration at the top. This exciting result could be evidence of a role for eATP in the Ca2+ uptake step, for an asymmetric gradient of eATP could direct the uptake preferentially along the bottom of the spore, a key step in initiating the trans-cell calcium current.

We want to confirm the microelectrode results with an independent method. Because firefly luciferase enzymes give off light in proportion to the [ATP] surrounding them, they have been used to report the [eATP] concentration in and outside of cells. We have constructed a reporter comprised of a luciferase protein with a cellulose-binding domain, expressed it in E. coli, and purified it using a published protocol. Currently, we are optimizing the protocol for cellulose-binding and luminescent imaging using the NightOWL imaging system (Berthold Technologies). The reporter will be applied to spores and used to assay for a top-to-bottom gradient of eATP in response to gravity, which would be an independent test of the ATP-specific microelectrode results described above.

We are also developing a method to visualize Ca2+ as it crosses the spore cell in the gravity-directed trans-cell current. Calcium Crimson (Life Technologies), a long-wavelength calcium indicator, is being tested for use as an intracellular calcium reporter in spores. The spores are mixed with the indicator and placed at different temperatures for varying periods of time based on the pollen loading protocol in Qu et al. (Qu et al., 2012). Spores have been screened for intracellular Calcium Crimson using an Axiovert 200 M Fluorescence Microscope (Zeiss), and initial experiments indicate a strong Ca2+ signal can be visualized. After this protocol is optimized, a confocal microscope will be used to assay changes in [Ca2+}cyt in response to uniform and unilateral ATP application.

Other accomplishments:

We have successfully re-established greenhouse procedures for growing Ceratopteris sporophytes and harvesting from them abundant spores that can be used for not only our experiments but also for experiments planned for SporeSat assays of the effects of different g-forces on the trans-cell calcium current.

We have completed 5 manuscripts (two primary research articles published, plus another one submitted, and two reviews to be submitted in July) related to our project.

Qu HY, Jiang XT, Shi ZB, Liu LM, Zhang SL (2012) Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia. Journal of Plant Research 125:185-­195